Novel probes for the detection of mycobacteria

ABSTRACT

Novel hybridisation assay probes and mixtures of such probes for detecting a target sequence of one or mycobacteria optionally present in a sample. The probes may suitable be directed to target sequences of mycobacterial rDNA, precursor rRNA, or rRNA, said probes being capable of forming detectable hybrids. The probes are in particular directed to mycobacterial rDNA, to precursor rRNA, or to 23S, 16S or 5S rRNA. The probes are useful for detecting the organisms in test samples such as sputum, laryngeal swabs, gastric lavage, bronchial washings, biopsies, aspirates, expectorates, body fluids (spinal, pleural, pericardial, synovial, blood, pus, bone marrow), urine, tissue sections as well as food samples, soil, air and water samples, and cultures thereof.

[0001] The present application claims priority under 35 USC 119(e) (1)from Provisional Application Nos. 60/028392 filed on Oct. 15, 1996,60/029595 filed on Oct. 23, 1996 and 60/045,962 fled on May 8, 1997.

[0002] The present invention relates to novel probes and to mixtures ofsuch probes, in addition to the design, construction and use of suchnovel probes or mixtures thereof for detecting a target sequence of oneor more mycobacteria, which probes are capable of detecting suchorganism(s) optionally present in a test sample, e.g. sputum, laryngealswabs, gastric lavage, bronchial washings, biopsies, aspirates,expectorates, body fluids (spinal, pleural, pericardial, synovial,blood, pus, bone marrow), urine, tissue sections as well as foodsamples, soil, air and water samples and cultures thereof. The inventionrelates in particular to novel probes and mixtures thereof for detectingthe presence of one or more mycobacteria of the Mycobacteriumtuberculosis Complex (MTC) and for detecting the presence of one or moremycobacteria other than mycobacteria of the Mycobacterium tuberculosisComplex (MOTT). The invention further relates to diagnostic kitscomprising one or more of such probes. The probes of the presentinvention are surprisingly able to penetrate the cell wall of themycobacteria, thus making possible the development of fast aneasy-performed in situ protocols.

BACKGROUND OF THE INVENTION

[0003] Tuberculosis is a very life-threatening and highly epidemicdisease which is caused by infection with Mycobacterium tuberculosis.Tuberculosis is presently the predominant infectious cause of morbidityand mortality world-wide, and is estimated to kill about three millionpeople annually. WHO estimates that the annual number of new cases oftuberculosis will increase from 7.5 million in 1990 to 10.2 million in2000, an escalation that will result in approximately 90 million newcases during this decade. It is furthermore estimated that 30 millionpeople will die from tuberculosis during the 1990s, which equals onequarter of preventable deaths among adults.

[0004] The prevalence of tuberculosis has been very high in the poorerparts of the world such as Asia, Africa and South-America, but in recentyears an increase has also been observed in industrialised countries.This appears to be due to an interaction of various factors includingi.a. patterns of migration, poorly organised tuberculosis programmes andnutrition problems. Furthermore, a serious threat will arise from theemergence of new strains that are drug resistant or worse, multi-drugresistant.

[0005] Mycobacteria are often divided into tuberculous mycobacteria,i.e. mycobacteria of the Mycobacterium tuberculosis Complex (MTC), andnon-tuberculous mycobacteria, i.e. mycobacteria other than those of theMycobacterium tuberculosis Complex (MOTT). The MTC group comprises apartfrom M. tuberculosis, M. bovis, M. africanum and M. microti.Mycobacteria of the MOTT group are not normally pathogenic to healthyindividuals but may cause disease in immunocompromised individuals, e.g.individuals infected with HIV. Clinical relevant mycobacteria of theMOTT group are in particular M. avium, M. intracellulare, M. kansasiiand M. gordonae, but also M. scrofulaceum, M. xenopi and M. fortuitum.

[0006]M. avium and M. intracellulare together with M. paratuberculosisand M. lepraemurium constitute the Mycobacterium avium Complex (MAC).Extended with M. scrofulaceum, the group is named Mycobacteriumavium-intracellulare-scrofulaceum Complex (MAIS).

[0007] It is well-known that treatment of mycobacterial infections withantibiotics may lead to the emergence of drug resistant strains. Manyantibiotic drugs excert their effects by interfering with proteinsynthesis or with transcription. Studies of the molecular mechanismsunderlying certain antibiotic resistance phenotypes in clinicalmycobacterium isolates have revealed mutations in rRNA genes. Thedevelopment of resistance because of mutation(s) located in the rRNAgene is likely to occur since slow growing mycobacteria have only asingle rRNA operon. All mycobacteria populations comprise a minority ofdrug resistant mutants that have arisen by spontaneous mutation. Thesemutated mycobacteria do normally not survive particularly well, but,when single-drug therapy is offered as treatment, the drug susceptiblebacteria are killed, and only the resistant mutants will survive andmultiply, and, thus at some point, constitute the majority of themycobacterial population. The selection of drug resistant bacteria dueto inadequate drug therapy leads to a state of so-called “acquireddrug-resistance”. In contrast, “primary drug-resistance” is used tocharacterise a situation where drug-resistant mycobacteria can beisolated from a patient who has never been treated for mycobacterialinfection, and has become infected with drug-resistant mycobacteria froman individual suffering from infection with an acquired drug resistantbacterium.

[0008] Today, drug-resistance is determined primarily phenotypically byculturing clinical samples, in which presence of mycobacteria have beendemonstrated, in the presence of the individual drugs. This isunfortunately a very slow and time-consuming procedure as the result ofthe drug-resistance studies depends on the growth rate of themycobacteria, which are well-known to be slow. Thus, the result is notavailable until after several weeks.

[0009] Although the incidence of drug-resistance is, at least not yet,very common, it is nevertheless very important that resistant strainsare identified and eradicated. Therefore, it is of major importance tofind a reliable and rapidly performed method of diagnosingdrug-resistance.

[0010] Presently, the detection of mycobacteria by microscopy is themost prevalent method for diagnosis. The sample (e.g. an expectorate) isstained for the presence of acid-fast bacilli using e.g. Ziehl-Neelsenstaining. However, staining for acid-fast bacilli does not provide thenecessary information about the type of infection, only whether acidfast bacilli are present in the sample, and this is in itself notsufficient information for establishing a diagnosis. Samples positivefor acid fast bacilli, may subsequently be cultured in order to be ableto perform species identification.

[0011] Since Ziehi-Neelsen staining cannot be used to determine whetherthe infection is caused by mycobacteria of the MTC group or mycobacteriaother than mycobacteria of the MTC group, a positive staining frequentlyleads to very costly isolation of all the patients with suspected M.tuberculosis infection as well as treatment with medicaments to whichthe patient may not even respond.

[0012] Since the sensitivity of acid fast staining is only approximately10⁴-10⁵ per ml smear negative samples should also be cultured asculture-based tests are sensitive, and as it may be possible to detect10-100 organisms per sample, but the result is not available before upto 8 weeks of culturing. Likewise, information about drug susceptibilityis not available until after 1-3 weeks of further testing.

[0013] Different solid or liquid media (Loewenstein Jensen slants andDubos broth) have traditionally been used for culturing ofmycobacteria-containing samples. Newer media include ESP Myco CultureSystem (Difco), MB/BacT (Organon Teknika), BacTec (Becton Dickinson) andMGIT (Becton Dickinson). These test media are based on colourmetric orfluorometric detection of carbon dioxide or oxygen produced bymycobacterial metabolism, and adapted to automated systems for largescale testing.

[0014] Species identification is presently carried out followingculturing using traditional biochemical methods or probe hybridisationassays (e.g. AccuProbe by Gen-Probe Inc., USA). There is, therefore, anincreasing need for means allowing a more rapid distinction betweenmycobacteria of the MTC group and mycobacteria other than those of theMTC group, and for further species identification of those especiallymycobacteria other than those of the MTC group.

[0015] A number of new attempts to replace the culture-based methodsrelies on molecular amplification technology. Several methods haveemerged, among them the polymerase chain reaction (PCR), the ligasechain reaction and transcription mediated amplification. The basicprinciple of amplification methods is that a specific nucleic acidsequence of the mycobacteria is amplified to increase the copy number ofthe specific sequence to a level where the amplicon may be detectable.In principle, the methods offers the possibility of detecting only onetarget sequence, thus, in principle, making detection of mycobacteriapresent at low levels possible. However, it has become clear that thetarget amplification methods cannot replace culture-based methods asonly samples which are positive by staining for acid fast bacilli (AFB)give a satisfactory sensitivity. Furthermore, specific problems existfor each method. The PCR method may give false negative results due tothe presence of inhibitors such as haemoglobin. Another problem arisesfrom cross-contamination of negative specimens and/or reagents withamplified nucleic acid present in the laboratory environment leading tofalse positive results. A disadvantage is that costly reagents areneeded for performing these tests. Furthermore, specialisedinstrumentation is required, making these tests mainly useful in largespecialised laboratories, and generally not applicable in smallerclinical laboratories.

[0016] Nucleic acid probes for detecting rRNA of mycobacteria have beendescribed in for example U.S. Pat. No. 5,547,842, EP-A 0 572 120 andU.S. Pat. No. 5,422,242.

[0017] Considering the perspective and impact the disease has, thedevelopment of rapid and preferably easy-performed and further economicfeasible diagnostic detection tests are of utmost importance and wouldbe a very valuable tool in the fight against the spread of tuberculosis.

[0018] Peptide nucleic acids are pseudo-peptides with DNA-bindingcapability. The compounds were first reported in the early nineties inconnection with a series of attempts to design nucleotide analoguescapable of hybridising, in a sequence-specific fashion, to DNA and RNA,cf. WO 92120702.

[0019] Hybridisation of peptide nucleic acid probes to DNA and to RNAhas been shown to obey the Watson-Crick base pairing rules, and peptidenucleic acid probes have been found to hybridise to a DNA or a RNAtarget with higher affinity and specificity than the nucleic acidcounterparts. These properties are ascribed to the uncharged, as opposedto the charged, structure of the peptide nucleic acid and DNA or RNAbackbones, respectively, and to the high conformational flexibility ofthe peptide nucleic acid molecules. These features—together with thedocumented stability of peptide nucleic acid towards a variety ofnaturally occurring nucleases and proteases that usually degrade DNA,RNA or proteins—invite for use of peptide nucleic acid probes asantisense therapeutic agents and opens potentially importantapplications in diagnostics.

SUMMARY OF THE INVENTION

[0020] The present invention relates to novel peptide nucleic acidprobes and to mixtures of such probes for detecting a target sequence ofone or more mycobacteria optionally present in a sample.

[0021] In a first aspect, the present invention relates to peptidenucleic acid probes for detecting a target sequence of one or moremycobacteria optionally present in a sample, said probes being capableof hybridising to a target sequence of mycobacterial rDNA, precursorrRNA or rRNA forming detectable hybrids. In another aspect, theinvention relates to peptide nucleic acid probes, said probe beingcapable of hybridising to a target sequence of mycobacterial rDNA,precursor rRNA, or 23S, 16S or 5S rRNA forming detectable hybrids

[0022] The peptide nucleic acid probes according to the presentinvention are capable of hybridising to a target sequence ofmycobacterial rDNA, precursor rRNA, or 23S, 16S or 5S rRNA formingdetectable hybrids, said target sequence being obtainable by

[0023] (a) comparing the nucleobase sequences of said mycobacterial rRNAor rDNA of one or more mycobacteria to be detected with thecorresponding nucleobase sequence of organism(s), in particular othermycobacteria, from which said one or more mycobacteria are to bedistinguished,

[0024] (b) selecting a target sequence of said rRNA or rDNA whichincludes at least one nucleobase differing from the correspondingnucleobase of the organism(s), in particular other mycobacteria, fromwhich said one or more mycobacteria are to be distinguished, and

[0025] (c) determining the capability.of said probe to hybridise to theselected target sequence to form detectable hybrids.

[0026] Furthermore, the peptide nucleic acid probes according to theinvention are capable of hybridising to a target sequence ofmycobacterial rDNA, precursor rRNA or 23S, 16S or 5S rRNA formingdetectable hybrids, said probe being obtainable by

[0027] (a) comparing the nucleobase sequences of said mycobacterial rRNAor rDNA of one or more mycobacteria to be detected with thecorresponding nucleobase sequence of organism(s), in particular othermycobacteria, in particular other mycobacteria, from which said one ormore mycobacteria are to be distinguished,

[0028] (b) selecting a target sequence of said rRNA or rDNA whichincludes at least one nucleobase differing from the correspondingnucleobase of the organism(s), in particular other mycobacteria, fromwhich said one or more mycobacteria are to be distinguished,

[0029] (c) synthesising said probe, and

[0030] (d) determining the capability of said probe to hybridise to theselected target sequence to form detectable hybrids.

[0031] In a further aspect, the invention relates to novel peptidenucleic acid probes for detecting a target sequence of one or moremycobacteria of the Mycobacterium tuberculosis Complex (MTC), or fordetecting a target sequence of one or more mycobacteria other thanmycobacteria of the Mycobacterium tuberculosis Complex (MOTT) optionallypresent in a sample, which probes comprises from 6 to 30 polymerisedpeptide nucleic acid moieties, said probe being capable of hybridisingto a target sequence of mycobacterial rDNA, precursor rRNA or 23S, 16Sor 5S rRNA forming detectable hybrids. Suitable probes are those offormula (I) comprising from 10 to 30 polymerised moieties of formula (I)

[0032] wherein each X and Y independently designate O or S,

[0033] each Z independently designates O, S, NR¹, or C(R¹)₂, whereineach R¹ independently designate H, C₁₋₆ alkyl, C₁₋₆ alkenyl, C₁₋₆alkynyl,

[0034] each R², R³ and R⁴ designate independently H, the side chain of anaturally occurring amino acid, the side chain of a non-naturallyoccurring amino acid, C₁₋₄ alkyl, C₁₋₄ alkenyl or C₁₋₄ alkynyl, or afunctional group, each Q independently designates a naturally occurringnucleobase, a non-naturally occurring nucleobase, an intercalator, anucleobase-binding group, a label or H,

[0035] with the proviso that the probe comprising such subsequence iscapable of forming detectable hybrids with the target sequence of saidmycobacterial rDNA, precursor rRNA or 23S, 16S or 5S rRNA.

[0036] Suitable probes for detecting a target sequence of 23S rRNA ofone or more mycobacteria of the Mycobacterium tuberculosis Complex (MTC)optionally present in a sample comprise from 10 to 30 polymerisedmoieties of formula (I) as defined above, with the proviso that the Qsof adjacent moieties are selected so as to form a sequence of which asubsequence includes at least one nucleobase that is complementary to anucleobase of M. tuberculosis 23S rRNA differing from the correspondingnucleobase of at least M. avium located within the following domains

[0037] Positions 149-158 in FIG. 1A,

[0038] Positions 220-221 in FIG. 1A,

[0039] Positions 328-361 in FIG. 1A and FIG. 1B,

[0040] Positions 453-455 in FIG. 1B.

[0041] Positions 490-501 in FIG. 1B,

[0042] Positions 637-660 in FIG. 1C,

[0043] Positions 706-712 in FIG. 1D,

[0044] Positions 762-789 in FIG. 1D,

[0045] Position 989 in FIG. 1D,

[0046] Positions 1068-1072 in FIG. 1D,

[0047] Position 1148 in FIG. 1E,

[0048] Positions 1311-1329 in FIG. 1E,

[0049] Positions 1361-1364 in FIG. 1F,

[0050] Position 1418 in FIG. 1F,

[0051] Positions 1563-1570 in FIG. 1F,

[0052] Positions 1627-1638 in FIG. 1G.

[0053] Positions 1675-1677 in FIG. 1G.

[0054] Position 1718 in FIG. 1G,

[0055] Positions 1734-1740 in FIG. 1H,

[0056] Positions 1967-1976 in FIG. 1H,

[0057] Positions 2403-2420 in FIG. 1H,

[0058] Positions 2457-2488 in FIG. 1I,

[0059] Positions 2952-2956 in FIG. 1I,

[0060] Positions 2966-2969 in FIG. 1J,

[0061] Positions 3000-3003 in FIG. 1J or

[0062] Positions 3097-3106 in FIG. 1J,

[0063] and further with the proviso that the probe comprising suchsubsequence is capable of forming detectable hybrids with a targetsequence of said mycobacterial 23S rRNA.

[0064] Suitable probes for detecting a target sequence of 16S rRNA ofone or more mycobacteria of the Mycobacterium tuberculosis Complex (MTC)optionally present in a sample comprise from 10 to 30 polymerisedmoieties of formula (I) as defined above, with the proviso that the Qsof adjacent moieties are selected so as to form a sequence of which asubsequence includes at least one nucleobase that is complementary to anucleobase of M. tuberculosis 16S rRNA differing from the correspondingnucleobase of at least M. avium located within the following domains

[0065] Positions 76-79 in FIG. 2A,

[0066] Positions 98-101 in FIG. 2A,

[0067] Positions 135-136 in FIG. 2A,

[0068] Positions 194-201 in FIG. 2B,

[0069] Positions 222-229 in FIG. 2B,

[0070] Position 242 in FIG. 2B,

[0071] Position 474 in FIG. 2C,

[0072] Positions 1136-1145 in FIG. 2C,

[0073] Positions 1271-1272 in FIG. 2C,

[0074] Positions 1287-1292 in FIG. 2D,

[0075] Position 1313 in FIG. 2D, or

[0076] Position 1334 in FIG. 2D,

[0077] and further with the proviso that the probe comprising suchsubsequence is capable of forming detectable hybrids with a targetsequence of said mycobacterial 16S rRNA.

[0078] Suitable probes for detecting a target sequence of 5S rRNA of oneor more mycobacteria of the Mycobacterium tuberculosis Complex (MTC)optionally present in a sample comprise from 10 to 30 polymerisedmoieties of formula (I) as defined above, with the proviso that the Qsof adjacent moieties are selected so as to form a sequence of which asubsequence includes at least one nucleobase that is complementary to anucleobase of M. tuberculosis 5S rRNA differing from the correspondingnucleobase of at least M. avium located within the following domain

[0079] Positions 86-90 in FIG. 3

[0080] and further with the proviso that the probe comprising suchsubsequence is capable of forming detectable hybrids with a targetsequence of said mycobacterial 5S rRNA.

[0081] In a preferred aspect, the invention relates to peptide nucleicacid probes for detecting a target sequence of 23S or 16S rRNA of one ormore mycobacteria of the Mycobacterium tuberculosis Complex (MTC)optionally present in a sample comprising from 10 to 30 polymerisedmoieties of formula (I) as defined above, with the proviso that the Qsof adjacent moieties are selected so as to form a sequence of which asubsequence includes at least one nucleobase that is complementary to anucleobase of M. tuberculosis 23S or 16S rRNA differing from thecorresponding nucleobase of at least M. avium located within thefollowing domains

[0082] Positions 149-158 in FIG. 1A,

[0083] Positions 328-361 in FIG. 1A and FIG. 1B,

[0084] Positions 490-501 in FIG. 1B,

[0085] Positions 637-660 in FIG. 1C,

[0086] Positions 762-789 in FIG. 1D,

[0087] Positions 1068-1072 in FIG. 1D,

[0088] Positions 1311-1329 in FIG. 1E,

[0089] Positions 1361-1364 in FIG. 1F,

[0090] Positions 1563-1570 in FIG. 1F,

[0091] Positions 1627-1638 in FIG. 1G,

[0092] Positions 1734-1740 in FIG. 1H,

[0093] Positions 2457-2488 in FIG. 1I,

[0094] Positions 2952-2956 in FIG. 1I,

[0095] Positions 3097-3106 in FIG. 1J,

[0096] Positions 135-136 in FIG. 2A, or

[0097] Positions 1287-1292 in FIG. 2D,

[0098] and further with the proviso that the probe comprising suchsubsequence is capable of forming detectable hybrids with a targetsequence of said mycobacterial 23S or 16S rRNA.

[0099] In a further embodiment, the present invention relates to peptidenucleic acid probes for detecting a target sequence of 23S rRNA of oneor more mycobacteria other than mycobacteria of the Mycobacteriumtuberculosis Complex (MOTT) optionally present in a sample comprisingfrom 10 to 30 polymerised moieties of formula (I) as defined above, withthe proviso that the Qs of adjacent moieties are selected so as to forma sequence of which a subsequence includes at least one nucleobase thatis complementary to a nucleobase of M. avium 23S rRNA differing from thecorresponding nucleobase of at least M. tuberculosis located within thefollowing domains

[0100] Positions 99-101 in FIG. 4A,

[0101] Position 183 in FIG. 4A,

[0102] Positions 261-271 in FIG. 4A,

[0103] Positions 281-284 in FIG. 4B,

[0104] Positions 290-293 in FIG. 4B,

[0105] Positions 327-335 in FIG. 4B,

[0106] Positions 343-357 in FIG. 4B,

[0107] Positions 400-405 in FIG. 4B and FIG. 4C,

[0108] Positions 453-462 in FIG. 4C,

[0109] Positions 587-599 in FIG. 4C,

[0110] Positions 637-660 in FIG. 4D,

[0111] Positions 704-712 in FIG. 4D,

[0112] Positions 763-789 in FIG. 4E,

[0113] Positions 1060-1074 in FIG. 4E,

[0114] Positions 1177-1185 in FIG. 4E,

[0115] Positions 1259-1265 in FIG. 4F,

[0116] Positions 1311-1327 in FIG. 4F,

[0117] Positions 1345-1348 in FIG. 4F,

[0118] Positions 1361-1364 in FIG. 4G,

[0119] Positions 1556-1570 in FIG. 4G,

[0120] Positions 1608-1613 in FIG. 4H,

[0121] Positions 1626-1638 in FIG. 4H,

[0122] Positions 1651-1659 in FIG. 4H,

[0123] Positions 1675-1677 in FIG. 4H,

[0124] Positions 1734-1741 in FIG. 4H,

[0125] Positions 1847-1853 in FIG. 4I,

[0126] Positions 1967-1976 in FIG. 4I,

[0127] Positions 2006-2010 in FIG. 4I,

[0128] Positions 2025-2027 in FIG. 4I,

[0129] Positions 2131-2132 in FIG. 4J,

[0130] Positions 2252-2255 in FIG. 4J,

[0131] Positions 2396-2405 in FIG. 4J and FIG. 4K,

[0132] Positions 2416-2420 in FIG. 4K,

[0133] Positions 2474-2478 in FIG. 4K,

[0134] Position 2687 in FIG. 4K,

[0135] Position 2719 in FIG. 4K,

[0136] Position 2809 in FIG. 4L,

[0137] Positions 3062-2068 in FIG. 4L, or

[0138] Positions 3097-3106 in FIG. 4L,

[0139] and further with the proviso that the probe comprising suchsubsequence is capable of forming detectable hybrids with a targetsequence of said mycobacterial 23S rRNA.

[0140] The invention further relates to peptide nucleic acid probes fordetecting a target sequence of 16S rRNA of one or more mycobacteriaother than mycobacteria of the Mycobacterium tuberculosis Complex (MOTT)optionally present in a sample comprising from 10 to 30 polymerisedmoieties of formula (I) as defined above, with the proviso that the Qsof adjacent moieties are selected so as to form a sequence of which asubsequence includes at least one nucleobase that is complementary to anucleobase of M. avium 16S rRNA differing from the correspondingnucleobase of at least M. tuberculosis located within the followingdomains

[0141] Positions 135-136 in FIG. 5A,

[0142] Positions 472-475 in FIG. 5A,

[0143] Positions 1136-1144 in FIG. 5A,

[0144] Positions 1287-1292 in FIG. 5B,

[0145] Position 1313 in FIG. 5B, or

[0146] Position 1334 in FIG. 5B,

[0147] and further with the proviso that the probe comprising suchsubsequence is capable of forming detectable hybrids with a targetsequence of said mycobacterial 16S rRNA.

[0148] In a preferred embodiment, the invention relates to peptidenucleic acid probes for detecting a target sequence of 23S or 16S rRNAof one or more mycobacteria other than mycobacteria of the Mycobacteriumtuberculosis Complex (MOTT) optionally present in a sample, which probescomprise from 10 to 30 polymerised moieties of formula (I) as definedabove, with the proviso that the Qs of adjacent moieties are selected soas to form a sequence of which a subsequence includes at least onenucleobase that is complementary to a nucleobase of M. avium 23S or 16SrRNA differing from the corresponding nucleobase of at least M.tuberculosis located within the following domains

[0149] Positions 99-101 in FIG. 4A,

[0150] Positions 290-293 in FIG. 4B,

[0151] Positions 400-405 in FIG. 4B and FIG. 4C,

[0152] Positions 453-462 in FIG. 4C,

[0153] Positions 637-660 in FIG. 4D,

[0154] Positions 763-789 in FIG. 4E,

[0155] Positions 1311-1327 in FIG. 4F,

[0156] Positions 1361-1364 in FIG. 4G,

[0157] Positions 1734-1741 in FIG. 4H,

[0158] Positions 2025-2027 in FIG. 4I,

[0159] Positions 2474-2478 in FIG. 4K,

[0160] Positions 3062-2068 in FIG. 4L, or

[0161] Positions 1287-1292 in FIG. 5B,

[0162] and further with the proviso that the probe comprising suchsubsequence is capable of forming detectable hybrids with a targetsequence of said mycobacterial 23S or 16S rRNA.

[0163] In another embodiment, the present invention relates to peptidenucleic acid probes for detecting a target sequence of 23S, 16S or 5SrRNA of one or more mycobacteria of the Mycobacterium tuberculosisComplex (MTC) or for detecting a target sequence of 23S, 16S or 5S rRNAof one or more mycobacteria other than mycobacteria of the Mycobacteriumtuberculosis Complex (MOTT), in particular drug resistant mycobacteria,optionally present in a sample, which probes comprise from 10 to 30polymerised moieties of formula (I) as defined above, with the provisothat the Qs of adjacent moieties are selected so as to form a sequenceof which a subsequence includes at least one nucleobase that iscomplementary to a nucleobase that differs from the correspondingnucleobase of 23S, 16S or 5S rRNA of said one or more mycobacterialocated within the following domains

[0164] Positions 2568-2569 in FIG. 6,

[0165] Position 452 in FIG. 7,

[0166] Positions 473-477 in FIG. 7, or

[0167] Positions 865-866 in FIG. 7,

[0168] and further with the proviso that the probe comprising suchsubsequence is capable of forming detectable hybrids with the targetsequence of said mycobacterial 23S, 16S or 5S rRNA.

[0169] In preferred embodiments, the peptide nucleic acid probesaccording to the invention are those of formula (II), (III), or (IV)

[0170] wherein Z, R², R³, and R⁴, and Q is as defined above, and furtherwith the provisos defined above. In may especially be preferred that Zis NH, NCH₃ or O, each R², R³ and R⁴ independently designate H or theside chain of a naturally occurring amino acid, the side chain of anon-naturally occurring amino acid, or C₁₋₄ alkyl, and each Q is anaturally occurring nucleobase or a non-naturally occurring nucleobase.In a further preferred embodiment, Z is NH or O, and R² is H or the sidechain of Ala, Asp, Cys, Glu, His, HomoCys, Lys, Orn, Ser or Thr, and Qis a nucleobase selected from thymine, adenine, cytosine, guanine,uracil, iso-C and 2,6-diaminopurine. The peptide nucleic acid probes maysuitably be those of formula (V)

[0171] wherein R⁴ is H or the side chain of Ala, Asp, Cys, Glu, His,HomoCys, Lys, Orn, Ser or Thr, and Q is as defined above, and with theprovisos defined above.

[0172] Such peptide nucleic acid probes may further comprise one or morelabels and a mixture of such probes, which labels may be mutuallyidentical or different, which probes optionally may comprise one or morelinkers, and which probes may be mutually identical or different withthe provisos defined above.

[0173] For many applications, it is preferred that the nucleobasesequence of the peptide nucleic acid probes is substantiallycomplementary to the nucleobase sequence of the target sequence. Inpreferred embodiments, the nucleobase sequence of said probe iscomplementary to the nucleobase sequence of said target sequence.

[0174] Peptide nucleic acid probes for detecting a target sequence ofone or more mycobacteria of the Mycobacterium tuberculosis Complex (MTC)or for detecting a target sequence of one or more mycobacteria otherthan mycobacteria of the Mycobacterium tuberculosis Complex (MOTT) aresuitably those wherein the Qs of adjacent moieties are selected so as toform the following subsequences AGA TGC GGG TAG GAG (selected frompositions 149-158 in FIG. 1A), (Seq ID no 1) TGT TTT CTC CTC CTA(selected from positions 220-221 in FIG. 1A), (Seq ID no 2) ACT GCC TCTCAG CCG (selected from positions 328-361 in FIG. 1A and FIG. 10), (SeqID no 3) TGA TAC TAG GCA GGT (selected from positions 453-455 in FIG.1B), (Seq ID no 4) CGG ATT CAC AGC GGA (selected from positions 490-501in FIG. 1B), (Seq ID no 5) TCA CCA CCC TCC TCC (selected from positions637-660 in FIG. 1C), (Seq ID no 6) CCA CCC TCC TCC (selected frompositions 637-660 in FIG. 1C), (modified Seq ID no 6) TTA ACC TTG CGACAT (selected from positions 706-712 in FIG. 1C), (Seq ID no 7) ACT ATTCAC ACG CGC (selected from positions 762-789 in FIG. 1D), (Seq ID no 8)CTC CGC GGT GAA CCA (selected from position 989 in FIG. 1D), (Seq ID no9) GCT TTA CAC CAC GGC (selected from positions 1068-1072 in FIG. 1D),(Seq ID no 10) ACG CTT GGG GGC CTT (selected from position 1148 in FIG.1E), (Seq ID no 11) CCA CAC CCA CCA CAA (selected from positions1311-1329 in FIG. 1E), (Seq ID no 12) CCG GTG GCT TCG CTG (selected frompositions 1361-1364 in FIG. 1F), (Seq ID no 13) ACT TGC CTT GTC GCT(selected from position 1418 in FIG. 1F), (Seq ID no 14) GAT TCG TCA CGGGCG (selected from positions 1563-1570 in FIG. 1F), (Seq ID no 15) AACTCC ACA CCC CCG (selected from positions 1627-1638 in FIG. 1G), (Seq IDno 16) ACT CCA CAC CCC CGA (selected from positions 1627-1638 in FIG.1G), (Seq ID no 17) ACC CCT TCG CTT GAC (selected from positions1675-1677 in FIG. 1G), (Seq ID no 18) CCT GCC CCA GTG TTA (selected fromposition 1718 in FIG. 1G), (Seq ID no 19) CTC TCC CTA CCG GCT (selectedfrom positions 1734-1740 in FIG. 1H), (Seq ID no 20) GAT ATT CCG GTC CCC(selected from positions 1967-1976 in FIG. 1H), (Seq ID no 21) ACT CCGCCC CAA CTG (selected from positions 2403-2420 in FIG. 1H), (Seq ID no22) CTG TCC CTA AAC CCG (selected from positions 2457-2488 in FIG. 1I),(Seq ID no 23) TTC GAG GTT AGA TGC (selected from positions 2457-2488 inFIG. 1I), (Seq ID no 24) GTC CCT AAA CCC GAT (selected from positions2457-2488 in FIG. 1I), (Seq ID no 25) GGT GCA CCA GAG GTT (selected frompositions 2952-2956 in FIG. 1I), (Seq ID no 26) CTG GCG GGA CAA CTG(selected from positions 2966-2969 in FIG. 1J), (Seq ID no 27) TTA TCCTGA CCG AAC (selected from positions 3000-3003 in FIG. 1J), (Seq ID no28) GAC CTA TTG AAC CCG (selected from positions 3097-3106 in FIG. 1J),(Seq ID no 29) GAA GAG ACC TTT CCG (selected from positions 76-79 inFIG. 2A), (Seq ID no 30) CAC TCG AGT ATC TCC (selected from positions98-101 in FIG. 2A), (Seq ID no 31) ATC ACC CAC GTG TTA (selected frompositions 136-136 in FIG. 2A), (Seq ID no 32) GCA TCC CGT GGT CCT(selected from positions 194-201 in FIG. 2B), (Seq ID no 33) CAC AAG ACATGC ATC (selected from positions 194-201 in FIG. 2B), (Seq ID no 34) TAAAGC GCT TTC CAC (selected from positions 222-229 in FIG. 2B), (Seq ID no35) GCT CAT CCC ACA CCG (selected from position 242 in FIG. 2B), (Seq IDno 36) CCG AGA GAA CCC GGA (selected from position 474 in FIG. 2C), (SeqID no 37) AGT CCC CAC CAT TAC (selected from positions 1136-1145 in FIG.2C), (Seq ID no 38) AAC CTC GCG GCA TCG (selected from positions1271-1272 in FIG. 2C), (Seq ID no 39) GGC TTT TAA GGA TTC (selected frompositions 1287-1292 in FIG. 2D), (Seq ID no 40) GAC CCC GAT CCG AAC(selected from position 1313 in FIG. 2D), (Seq ID no 41) CCG ACT TCA CGGGGT (selected from position 1334 in FIG. 2D), (Seq ID no 42) CGG AGG GGCAGT ATC (selected from positions 86-90 in FIG. 3), (Seq ID no 43) GATCAA TGC TCG GTT (selected from positions 99-101 in FIG. 4A), (Seq ID no44) TTC CCC GCG TTA CCT (selected from position 183 in FIG. 4A), (Seq IDno 45) TTA GCC TGT TCC GGT (selected from positions 261-271 in FIG. 4A),(Seq ID no 46) GCA TGC GGT TTA GCC (selected from positions 281-284 inFIG. 4B), (Seq ID no 47) TAC CGG GTT GTC CAT (selected from positions290-293 in FIG. 4B), (Seq ID no 48) GTA GAG GTG AGA CAT (selected fromposition 327-335 and 343-357 in FIG. 4B), (Seq ID no 49) GCC GTC CCA GGCGAG (selected from positions 400-405 in FIG. 4B and FIG. 4C), (Seq ID no50) CTC GGG TGT TGA TAT (selected from positions 453-462 in FIG. 4C),(Seq ID no 51) ACT ATT TCA CTG GGT (selected from positions 587-599 inFIG. 4C), (Seq ID no 52) ACG CCA TCA CCC GAG (selected from positions637-660 in FIG. 4D), (Seq ID no 53) CGA GGT GTC GCT GAG (selected frompositions 704-712 in FIG. 4D), (Seq ID no 54) ACT ACA CCC CAA AGG(selected from positions 763-789 in FIG. 4E), (Seq ID no 55) CAC GCT TTTACA CCA (selected from positions 1060-1074 in FIG. 4E), (Seq ID no 56)GCG ACT ACA CAT CCT (selected from positions 1177-1185 in FIG. 4E), (SeqID no 57) CGG CGC ATA ATC ACT (selected from positions 1259-1265 in FIG.4F), (Seq ID no 58) CCA CAT CCA CCG TAA (selected from positions1311-1327 in FIG. 4F), (Seq ID no 59) CGC TGA ATG GGG GAC (selected frompositions 1345-1348 in FIG. 4F), (Seq ID no 60) GGA GCT TCG CTG AAT(selected from positions 1361-1364 in FIG. 4G), (Seq ID no 61) CGG TCACCC GGA GCT (selected from positions 1361-1364 in FIG. 4G), (Seq ID no62) GGA CGC CCA TAC ACG (selected from positions 1556-1570 in FIG. 4G),(Seq ID no 63) GAA GGG GAA TGG TCG (selected from positions 1608-1613 inFIG. 4H), (Seq ID no 64) AAT CGC CAC GCC CCC (selected from positions1626-1638 in FIG. 4H), (Seq ID no 65) CAG CGA AGG TCC CAC (selected frompositions 1651-1659 in FIG. 4H), (Seq ID no 66) GTC ACC CCA TTG CTT(selected from positions 1675-1677 in FIG. 4H), (Seq ID no 67) ATC GCTCTC TAC GGG (selected from positions 1734-1741 in FIG. 4H), (Seq ID no68) GTG TAT GTG CTC GCT (selected from positions 1847-1853 in FIG. 4I),(Seq ID no 69) ACG GTA TTC CGG GCC (selected from positions 1967-1976 inFIG. 4I), (Seq ID no 70) GGC CGA ATC CCG CTC (selected from positions2006-2010 in FIG. 4I), (Seq ID no 71) AAA CAG TCG CTA CCC (selected frompositions 2025-2027 in FIG. 4I), (Seq ID no 72) CCT TAC GGG TTA ACG(selected from positions 2131-2132 in FIG. 4J), (Seq ID no 73) GAG ACAGTT GGG AAG (selected from positions 2252-2255 in FIG. 4J), (Seq ID no74) TGG CGT CTG TGC TTC (selected from positions 2396-2405 in FIG. 4Jand FIG. 4K), (Seq ID no 75) CGA CTC CAC ACA AAC (selected frompositions 2416-2420 in FIG. 4K), (Seq ID no 76) GAT AAG GGT TCG ACG(selected from positions 2474-2478 in FIG. 4K), (Seq ID no 77) ATC CGTTGA GTG ACA (selected from position 2687 in FIG. 4K), (Seq ID no 78) CAGCCC GTT ATC CCC (selected from position 2719 in FIG. 4K), (Seq ID no 79)AAC CTT TGG GAC CTG (selected from position 2809 in FIG. 4L), (Seq ID no80) TAA AAG GGT GAG AAA (selected from positions 3062-3068 in FIG. 4L),(Seq ID no 81) GTC TGG CCT ATC AAT (selected from positions 3097-3106 inFIG. 4L), (Seq ID no 82) AGA TTG CCC ACG TGT (selected from positions135-136 in FIG. 5A), (Seq ID no 83) AAT CCG AGA AAA CCC (selected frompositions 472-475 in FIG. 5A), (Seq ID no 84) GCA TTA CCC GCT GGC(selected from positions 1136-1144 in FIG. 5B), (Seq ID no 85) TTA AAAGGA TCC GCT (selected from positions 1287-1292 in FIG. 5B), (Seq ID no86) AGA CCC CAA TCC GAA (selected from position 1313 in FIG. 5B), (SeqID no 87) GAC TCC GAG TTC ATG (selected from position 1334 in FIG. 5B),(Seq ID no 88) GTC TTT TCG TCC TGC (selected from positions 2568-2569 inFIG. 6), (Seq ID no 89) GTC TTA TCG TCC TGC (selected from positions2568 in FIG. 6), (Seq ID no 90) GTC TTC TCG TCC TGC (selected frompositions 2568 in FIG. 6), (Seq ID no 91) GTC TTG TCG TCC TGC (selectedfrom positions 2568 in FIG. 6), (Seq ID no 92) GTC TAT TCG TCC TGC(selected from positions 2568 in FIG. 6), (Seq ID no 93) GTC TCT TCG TCCTGC (selected from positions 2568 in FIG. 6), (Seq ID no 94) GTC TGT TCGTCC TGC (selected from positions 2568 in FIG. 6), (Seq ID no 95) TTG GCCGGT GCT TCT (selected from positions 452 in FIG. 7), (Seq ID no 96) TTGGCC GGT ACT TCT (selected from positions 452 in FIG. 7), (Seq ID no 97)TTG GCC GGT CCT TCT (selected from positions 452 in FIG. 7), (Seq ID no98) TTG GCC GGT TCT TCT (selected from positions 452 in FIG. 7), (Seq IDno 99) ACC GCG GCT GCT GGC (selected from positions 473-477 in FIG. 7),(Seq ID no 100) ACC GCG GCT ACT GGC (selected from positions 473 in FIG.7), (Seq ID no 101) ACC GCG GCT CCT GGC (selected from positions 473 inFIG. 7), (Seq ID no 102) ACC GCG GCT TCT GGC (selected from positions473 in FIG. 7), (Seq ID no 103) CGG CAG CTG GCA CGT (selected frompositions 474 in FIG. 7), (Seq ID no 104) CGG CCG CTG GCA CGT (selectedfrom positions 474 in FIG. 7), (Seq ID no 105) CGG CTG CTG GCA CGT(selected from positions 474 in FIG. 7), (Seq ID no 106) CGT ATT ACC GCAGCT (selected from positions 477 in FIG. 7), (Seq ID no 107) CGT ATT ACCGCC GCT (selected from positions 477 in FIG. 7), (Seq ID no 108) CGT ATTACC GCT GCT (selected from positions 477 in FIG. 7), (Seq ID no 109) TTCCTT TGA GTT TTA (selected from positions 865-866 in FIG. 7), (Seq ID no110) TTC CTT TAA GTT TTA (selected from positions 865 in FIG. 7), (SeqID no 111) TTC CTT TCA GTT TTA (selected from positions 865 in FIG. 7),(Seq ID no 112) TTC CTT TTA GTT TTA (selected from positions 865 in FIG.7), (Seq ID no 113) TTC CTT AGA GTT TTA (selected from positions 866 inFIG. 7), (Seq ID no 114) TTC CTT CGA GTT TTA (selected from positions866 in FIG. 7), (Seq ID no 115) TTC CTT GGA GTT TTA (selected frompositions 866 in FIG. 7), (Seq ID no 116) CAT GTG TCC TGT GGT (Seq ID no117) CGT CAG CCC GAG AAA (Seq ID no 118) CAC TAC ACA CGC TCG (Seq ID no119) TGG CGT TGA GGT TTC and (Seq ID no 120) AAC ACT CCC TTT GGA. (SeqID no 123)

[0175] In a preferred embodiment, such probes are those wherein the Qsof adjacent moieties are selected so as to form the followingsubsequences TCA CCA CCC TCC TCC (Seq ID no 6) CCA CCC TCC TCC (modifiedSeq ID no 6) ACT ATT CAC ACG CGC (Seq ID no 8) CCA CAC CCA CCA CAA (SeqID no 12) AAC TCC ACA CCC CCG (Seq ID no 16) ACT CCA GAG CCC CGA (Seq IDno 17) ACT CCG CCC CAA GTG (Seq ID no 22) CTG TCC CTA AAC CGG (Seq ID no23) TTC GAG GTT AGA TGC (Seq ID no 24) GTG CCT AAA CCC GAT (Seq ID no25) GAC CTA TTG AAC CCG (Seq ID no 29) GCA TCC CGT GGT CCT (Seq ID no33) CAC AAG ACA TGC ATC (Seq ID no 34) GGC TTT TAA GGA TTG (Seq ID no40) GAT CAA TGC TCG GTT (Seq ID no 44) CGA CTC CAC ACA AAC (Seq ID no76) GCA TTA CCC GCT GGC (Seq ID no 85) GTG TTA TCG TCC TGC (Seq ID no90) GTC TTC TCG TCC TGC (Seq ID no 91) GTG TTG TCG TCC TGC (Seq ID no92) GTC TAT TCG TCC TGC (Seq ID no 93) GTC TCT TCG TCC TGC (Seq ID no94) GTG TGT TCG TCC TGC (Seq ID no 95) AAC ACT CCC TTT GGA (Seq ID no123) CAT GTG TCC TGT GGT (Seq ID no 117) CGT CAG CCC GAG AAA (Seq ID no118) CAC TAC ACA CGC TCG, (Seq ID no 119) TGG CGT TGA GGT TTC (Seq ID no120)

[0176] In accordance herewith, the present invention relates to peptidenucleic acid probes selected from Lys(Flu)-Lys(Flu)-TGA CCA CCC TCCTCC-NH₂ (OK 446/modified Seq ID no 6) Lys(Flu)-Lys(Flu)-CCA CCC TCCTCC-NH₂ (OK 575/modified Seq ID no 6) Lys(Flu)-Lys(Flu)-ACT ATT CAC ACGCGC-NH₂ (OK 447/modified Seq ID no 8) Lys(Flu)-ACT ATT CAC ACG CGC-NH₂(OK 688/modified Seq ID no 8) Lys(Flu)-Lys(Flu)-CCA CAC CCA CCA CAA-NH₂(OK 448/modified Seq ID no 12) Lys(Flu)-Lys(Flu)-AAC TCC ACA CCC CCG-NH₂(OK 449/modified Seq ID no 16) Lys(Flu)-Lys(Flu)-ACT CCA CAC CCC CGA-NH₂(OK 309/modified Seq ID no 17) Lys(Flu)-Lys(Flu)-ACT CCG CCC CAA CTG-NH₂(OK 450/modified Seq ID no 22) Lys(Flu)-Lys(Flu)-CTG TCC CTA AAC CCG-NH₂(OK 305/modified Seq ID no 23) Lys(Flu)-Lys(Flu)-TTC GAG GTT AGA TGC-NH₂(OK 306/modified Seq ID no 24) Lys(Flu)-TTC GAG GTT AGA TGC-NH₂ (OK682/modified Seq ID no 24) Lys(Flu)-Lys(Flu)-GTC CCT AAA CCC GAT-NH₂ (OK307/modified Seq ID no 25) Lys(Flu)-GTC CCT AAA CCC GAT-NH₂ (OK654/modified Seq ID no 25) Lys(Flu)-GAC CTA TTG AAC CCG-NH₂ (OK660/modified Seq ID no 29) Lys(Flu)-Lys(Flu)-Gly-GCA TCC CGT GGT CCT-NH₂(OK 223/modified Seq ID no 33) Lys(Flu)-Lys(Flu)-CAC AAG ACA TGC ATC-NH₂(OK 310/modified Seq ID no 34) Lys(Flu)-CAC AAG ACA TGC ATC-NH₂ (OK655/modified Seq ID no 34) Lys(Flu)-GGC TTT TAA GGA TTC-NH₂ (OK689/modified Seq ID no 40) Lys(Rho)-GGC TTT TAA GGA TTC-NH₂ (OK689/modified Seq ID no 40) Flu-β-Ala-β-Ala-GAT CAA TGC TCG GTT-NH₂ (OK624/modified Seq ID no 44) Flu-β-Ala-β-Ala-CGA CTC CAC ACA AAC-NH₂ (OK612/modified Seq ID no 76) Flu-β-Ala-β-Ala-GCA TTA CCC GCT GGC-NH₂ (OK623/modified Seq ID no 65) Lys(Flu)-GTC TTT TCG TCC TGC-NH₂ (OK745/modified Seq ID no 89) Lys(Rho)-GTC TTA TCG TCC TGC-NH₂ (OK746/modified Seq ID no 90) Lys(Rho)-GTC TTC TCG TCC TGC-NH₂ (OK746/modified Seq ID no 91) Lys(Rho)-GTC TTG TCG TCC TGC-NH₂ (OK746/modified Seq ID no 92) Lys(Rho)-GTC TAT TCG TCC TGC-NH₂ (OK747/modified Seq ID no 93) Lys(Rho)-GTG TCT TCG TCC TGC-NH₂ (OK747/modified Seq ID no 94) Lys(Rho)-GTC TGT TCG TCC TGC-NH₂ (OK747/modified Seq ID no 95) Lys(Flu)-AAC ACT CCC TTT GGA-NH₂ (OK749/modified Seq ID no 123)

[0177] wherein Flu denotes a 5-(and 6)-carboxyfluoroescein label and Rhodenotes a rhodamine label.

[0178] In a further aspect, the invention relates to the use of peptidenucleic acid probes as defined above or a mixture thereof for detectinga target sequence of one or more mycobacteria optionally present in asample. In particular, the invention relates to the use of a peptidenucleic acid probe or a mixture thereof for detecting a target sequenceof one or more mycobacteria of the Mycobacterium tuberculosis Complex(MTC), in particular a target sequence of M. tuberculosis, and furtherto the use of peptide nucleic acid probes or a mixture thereof fordetecting a target sequence of one or more mycobacteria other thanmycobacteria of the Mycobacterium tuberculosis Complex (MOTT), inparticular a target sequence of one or more mycobacteria of theMycobacterium avium Complex.

[0179] The invention further relates to a method for detecting a targetsequence of one or more mycobacteria optionally present in a samplecomprising

[0180] (1) contacting any rRNA or rDNA present in said sample with oneor more peptide nucleic acid probes as defined above or a mixturethereof under conditions, whereby hybridisation takes place between saidprobe(s) and said rRNA or rDNA, and

[0181] (2) observing or measuring any formed detectable hybrids, andrelating said observation or measurement to the presence of a targetsequence of one or more mycobacteria in said sample.

[0182] In particular, the invention relates to a method for detecting atarget sequence of one or more mycobacteria of the Mycobacteriumtuberculosis Complex (MTC), in particular a target sequence of M.tuberculosis, or to a method for detecting a target sequence of one ormore mycobacteria other than mycobacteria of the Mycobacteriumtuberculosis Complex (MOTT). In preferred embodiments, the hybridisationtakes place in situ, or takes place in vitro. In an embodiment, a signalamplifying system is used for measuring the resulting hybridisation. Itis further preferred that the sample is a sputum sample.

[0183] Furthermore, the invention relates to kits for detecting a targetsequence of one or more mycobacteria, in particular a target sequence ofone or more mycobacteria of the Mycobacterium tuberculosis Complex(MTC), and in particular a target sequence of M. tuberculosis, and/orfor detecting a target sequence of one or more mycobacteria other thanmycobacteria of the Mycobacterium tuberculosis Complex (MOTT), inparticular a target sequence of one or more mycobacteria of theMycobacterium avium Complex (MAC), which kit comprise at least onepeptide nucleic acid probe as defined above, and optionally a detectionsystem with at least one detecting reagent. In one embodiment thereof,the kit further comprises a solid phase capture system.

BRIEF DESCRIPTION OF THE FIGURES

[0184] Alignments of rDNA sequences of M. tuberculosis (as arepresentative of the MTC group) and important closely related speciesthereto, including M. avium (as a representative of the mycobacteriaother than those of the MTC group) and important closely related speciesthereto for the 23S, 16S and/or 5S rRNA genes have been made (FIGS.1A-1J, 2A-2D, 3, 4A-4L and 5A-B). The alignment for M. bovis and M.intracellulare are partly based on public available sequences and partlyon sequences obtained by sequencing performed at DAKO A/S.

[0185] Alignment for the MTC group (23S rDNA)

[0186] FIGS. 1A-1J show alignments of 23S rDNA sequences of M.tuberculosis (GenBank entry GB:MTCY130, accession number Z73902), M.avium (GenBank entry GB:MA23SRNA, accession number X74494), M.paratuberculosis (GenBank entry GB:MPARRNA, accession number X74495), M.phlei (GenBank entry GB:MP23SRNA, accession number X74493), M. leprae(GenBank entry GB:ML5S23S, accession number X56657), M. gastri (GenBankentry GB:MG23SRRNA, accession numberZ17211), M. kansasii (GenBank entryGB:MK23SRRNA, accession number Z17212), and M. smegmatis (GB:MS16S23S5,accession number Y08453). Preferred peptide nucleic acid probes shouldenclose at least one nucleobase complementary to a nucleobase of M.tuberculosis 23S rRNA within positions 149-158, 220-221, 328-361,453-455,490-501, 637-660, 706-712, 762-789, 989, 1068-1072, 1148,1311-1329, 1361-1364, 1418, 1563-1570, 1627-1638, 1675-1677,1718,1734-1740, 1967-1976, 2403-2420, 2457-2488, 2952-2956, 2966-2969,3000-3003, and 3097-3106 of the alignment (indicated by heavy frames).Differences between the sequences of M. avium, M. phlei, M. leprae, M.paratuberculosis, M. gastri and M. kansasii and that of M. tuberculosisin the alignment are indicated by light frames.

[0187] Alignment for the MTC group (16S rDNA)

[0188] FIGS. 2A-2D show alignments of 16S rDNA sequences of M.tuberculosis (GenBank entry GB:MTU16SRN, accession number X52917), M.bovis (GenBank entry GB:MSGTGDA, accession number M20940), M. avium(GenBank entry GB:MSGRRDA, accession number M61673), M. intracellulare(GenBank entry GB:MIN16SRN, accession number X52927), M.paratuberculosis (GenBank entry GB:MSGRRDH, accession number M61680), M.scrofulaceum (GenBank entry GB:MSC16SRN, accession number X52924), M.leprae (GenBank entry GB:MLEP16S1, accession number X55587), M. kansasii(GenBank entry GB:MKRRN16, accession number X15916), M. gastri (GenBankentry GB:MGA16SRN, accession number X52919), M. gordonae (GenBank entryGB:MSGRR16SI, accession number M29563) and M. marinum (GenBank entryGB:MMA16SRN, accession number X52920). Preferred peptide nucleic acidprobes should enclose at least one nucleobase complementary to anucleobase of M. tuberculosis 16S rRNA within positions 76-79, 98-101,135-136, 194-201,222-229, 242, 474, 1136-1145, 1271-1272, 1287-1292,1313, and 1334 of the alignment (indicated by heavy frames). Differencesbetween the sequences of M. bovis, M. avium, M. intracellulare, M.paratuberculosis, M. scrofulaceum, M. Ieprae, M. kansasii, M. gastri, M.gordonae and M. marinum, and that of M. tuberculosis in the alignmentare indicated by light frames.

[0189] Alignment for the MTC group (5S rDNA)

[0190]FIG. 3 shows alignments of 5S rDNA sequences of M. tuberculosis(GenBank entry GB:MTDNA16S, accession number x75601), M. bovis (GenBankentry GB:MBRRN5S, accession number X05526), M. phlei (GenBank entryGB:MP5SRRNA, accession number X55259), M. Ieprae (GenBank entryGB:ML5S23S, accession number X56657), and M. smegmatis (GenBank entryGB:MS16S23S5, accession number Y08453). Preferred peptide nucleic acidprobes should enclose at least one nucleobase complementary to anucleobase of M. tuberculosis 5S rRNA within positions 86-90 of thealignment (indicated by heavy frame). Differences between the sequencesof M. bovis, M. phlei, M. leprae, M. smegmatis and M. luteus and that ofM. tuberculosis in the alignment are indicated by light frames.

[0191] Alignment for Mycobacteria other than those of the MTC group (23SrDNA)

[0192] FIGS. 4A-4L show alignments of 23S rDNA sequences of M. avium(GenBank entry GB:MA23SRNA, accession number X74494), M.paratuberculosis (GenBank entry GB:MPARRNA, accession number X74495), M.tuberculosis (GenBank entry GB:MTCY130, accession number Z73902), M.phlei (GenBank entry GB:MP23SRNA, accession number X74493), M. leprae(GenBank entry GB:ML5S23S, accession number X56657), M. gastri (GenBankentry GB:MG23SRRNA, accession number Z17211), M. kansasii (GenBank entryGB:MK23SRRNA, accession number Z17212), and M. smegmatis (GB:MS16S23S5,accession number Y08453). Preferred peptide nucleic acid probes shouldenclose at least one nucleobase complementary to a nucleobase of M.avium 23S rRNA within positions 99-101, 183, 261-271, 281-284, 290-293,327-335, 343-357, 400-405, 453-462, 587-599, 637-660, 704-712, 763-789,1060-1074, 1177-1185, 1259-1265, 1311-1327, 1345-1348. 1361-1364,1556-1570, 1608-1613, 1626-1638, 1651-1659, 1675-1677, 1734-1741,1847-1853, 1967-1976, 2006-2010, 2025-2027, 2131-2232, 2252-2255,2396-2405, 2416-2420, 2474-2478, 2687, 2719, 2809, 3062-3068, and3097-3106 of the alignment (indicated by heavy frames). Differencesbetween the sequences of M. paratuberculosis, M. tuberculosis, M. phlei,M. leprae, M. gastri, M. kansasii, and M. smegmatis and that of M. aviumin the alignment are indicated by light frames.

[0193] Alignment for Mycobacteria other than those of the MTC group (16SrDNA)

[0194] FIGS. 5A-5B show alignments of 16S rDNA sequences of M. avium(GenBank entry GB-MSGRRDA, accession number M61673), M. intracellulare(GenBank entry GB:MIN16SRN, accession number X52927), M.paratuberculosis (GenBank entry GB:MSGRRDH, accession number M61680), M.scrofulaceum (GenBank entry GB:MSC16SRN, accession number X52924), M.tuberculosis (GenBank entry GB:MTU16SRN, accession number X52917), M.bovis (GenBank entry GB:MSGTGDA, accession number M20940), M. leprae(GenBank entry GB:MLEP16S1, accession number X55587), M. kansasii(GenBank entry GB:MKRRN16, accession number X15916), and M. gastri(GenBank entry GB:MGA16SRN, accession number X52919), M. gordonae(GenBank entry GB:MSGRR16SI, accession number M29563), and M. marinum(GenBank entry GB:MMA16SRN, accession number X52920). Preferred peptidenucleic acid probes should enclose at least one nucleobase complementaryto a nucleobase of M. avium 16S rRNA within positions 135-136, 472-475,1136-1144, 1287-1292, 1313, and 1334 of the alignment (indicated byheavy frames). Differences between the sequences of M. intracellulare,M. paratuberculosis, M. scrofulaceum, M. tuberculosis, M. bovis, M.leprae, M. kansasii, and M. gastri and that of M. avium in the alignmentare indicated by light frames.

[0195] Drug-resistance

[0196]FIG. 6 shows a partial M. avium 23S rDNA sequence includingpositions 2550 to 2589 of GenBank entry X74494. Bases in positions wheredeviations from the wild-type sequence have been correlated withmacrolide-resistance are framed. Positions 2568 and 2569 in the figurecorrespond to positions 2058 and 2059, respectively, of E.coli 23S rRNA.

[0197]FIG. 7 shows a partial M. tuberculosis 16S rDNA sequence includingpositions 441 to 491 and B43 to 883 of GenBank entry X52917. Bases inpositions where deviations from the wild-type sequence have beencorrelated with resistance to streptomycin are framed. Positions 452,473, 474, 477, 865, and 866 in the figure correspond to positions 501,522, 523, 526, 912, and 913, respectively, of E.coli 16S rRNA.

SPECIFIC DESCRIPTION

[0198] Mycobacteria are characterised by a complex cell wall whichcontains myolic acids, complex waxes and unique glycolipids. It isgenerally recognised by those skilled in the art that this wall providesmycobacteria with extreme resistance to chemical and physical stress ascompared to other bacteria, and, accordingly, makes them very difficultto penetrate and lyse. The low permeability of the cell wall isconsidered to be the main reason for the fact that only very few drugsare effective in the treatment of tuberculosis and other mycobacterialinfections. As described in U.S. Pat. No. 5,582,985, the wall appearsfurther to prevent penetration by nucleic acid probes. Even with shortprobes (shorter than 30 nucleic acids), specific staining is low oroften non-existent. Protocols that allow DNA probes to be used for insitu hybridisation to mycobacterial species are described in U.S. Pat.No. 5,582,985. However, these protocols require dewaxing of themycobacterial cell wall with xylene and further enzymatic treatmentprior to the hybridisation step in order to make the mycobacterial cellwall permeable to the DNA probes.

[0199] The problems set forth above have surprisingly been completelysolved by the use of peptide nucleic acid probes. It has, surprisingly,been found that the peptide nucleic acid probes are able to penetratethe cell wall of the mycobacteria, and furthermore that this is takingplace rapidly. The person skilled in the art would arrive at theconviction that it would be necessary to heavily treat the mycobacteriabefore hybridisation is carried out. Thus, based on the available priorart, there is a strong prejudice against carrying out hybridisationwithout prior destruction of the mycobacterial cell wall. The inventorshave shown that this is indeed and unexpectedly possible. It has beendemonstrated that the probes of the present invention are able tohybridise to mycobacterial precursor rRNA and rRNA without harshtreatment of the mycobacterial cells, thus avoiding a risk ofinterfering with the morphology of the cells. Using the present probes,specific and easy detection and, subsequently, diagnosis of tuberculosisand other mycobacterial infections are thus possible.

[0200] The present invention provides novel probes for use in rapid andspecific, sensitive hybridisation based assays for detecting a targetsequence of one or more mycobacteria, which target sequence is locatedin the mycobacterial rDNA, precursor rRNA, or in the 23S, 16S or 5SrRNA. The probes to be used in accordance with the present invention arepeptide nucleic acid probes. Peptide nucleic acids are non-naturallyoccurring polyamides or polythioamides which can bind to nucleic acids(DNA and RNA). Such compounds are described in e.g. WO 92/20702.

[0201] We have identified suitable variable regions of the targetnucleic acid by comparative analysis of generally available rDNAsequences and sequences obtained by sequencing as described above.Computers and computer programs, which have been used for the purposesdisclosed herein, are commercially available. From such alignments,possibly suitable probes can be identified. The alignments are thus auseful guideline for designing probes with desired characteristics.

[0202] When designing the probes, due regard should be taken to theassay conditions under which the probes are to be used. Stringency ischosen so as to maximise the difference in stability between the hybridformed with the target nucleic acid and that formed with the non-targetnucleic acid. It will typically be necessary to choose high stringencyconditions for probes where the specificity depends on only one mismatchto non-target sequences The more mismatches to non-target sequences, theless demand for high stringency conditions.

[0203] Furthermore, probes should be designed so as to minimise thestability of probe-non-target nucleic acid hybrids. This may beaccomplished by minimising the degree of complementarity to non-targetnucleic acid, i.e. by designing the probe to span as many destabilisingmismatches as possible, and/or to include as many additions/deletionsrelative to the target sequence as possible. Whether a probe is usefulfor detecting a particular mycobacterial species depends to some degreeon the difference between the thermal stability of probe-target hybridsand probe:non-target hybrids. For rRNA targets, however, the secondarystructure of the region of the rRNA molecule in which the targetsequence is located may also be of importance. The secondary structureof a probe should also be taken into consideration. Probes should bedesigned so as to minimise their proclivity to form hairpins,self-dimers, and pair-dimers if a mixture of two or more probes is used.

[0204] Mismatching bases in hybrids formed between peptide nucleic acidprobes and nucleic acids result in a higher thermal instability thanmismatching bases in nucleic acid duplexes of the same sequences. Thus,the peptide nucleic acid probes exhibit a greater specificity for agiven target nucleic acid sequence than a traditional nucleic acidprobe, which is seen as a greater difference in T_(m) values forprobe-target hybrids and probe-non-target hybrids. The sensitivity andspecificity of a peptide nucleic acid probe will also depend on thehybridisation conditions used.

[0205] The primary concern regarding the length of the peptide nucleicacid probes is the warranted specificity, i.e. which length providessufficient specificity for a particular application. The optimal lengthof a peptide nucleic acid probe comprising a particular site withdifferences in base composition, e.g. among selected regions ofmycobacterial rRNA, is a compromise between the general pattern thatlonger probes ensure specificity and shorter probes ensure that thedestabilising differences in base composition constitute a greaterportion of the probe Also, due regard must be paid to the conditionsunder which the probes are to be used.

[0206] Peptide nucleic acid sequences are written from the N-terminalend of the sequence towards the C-terminal end. A free (unsubstituted)N-terminal end or an N-terminal end terminating with an amino acid isindicated as H, and a free C-terminal end is indicated as NH₂ (acarboxamide group). Peptide nucleic acids are capable of hybridising tonucleic acid sequences in two orientations, namely in antiparallelorientation and in parallel orientation. The peptide nucleic acid issaid to hybridise in the antiparallel orientation when the N-terminalend of the peptide nucleic acid is facing the 3′ end of the nucleic acidsequence, and to hybridise in the parallel orientation when. theC-terminal end of the peptide nucleic acid is facing the 5′ end of thenucleic acid sequence. In most applications, hybridisation in theantiparallel orientation is preferred as the hybridisation in theparallel orientation takes place rather slowly and as the formedduplexes are not as stable as the duplexes having antiparallel strands.Triplex formation with a stoichiometry of two peptide nucleic acidstrands and one nucleic acid strand may occur if the peptide nucleicacid has a high pyrimidine content. Such triplexes are very stable, andprobes capable of forming triplexes may thus be suitable for certainapplications.

[0207] Mainly because the peptide nucleic acid strand is uncharged, apeptide nucleic acid-nucleic acid-duplex will have a higher T_(m) thanthe corresponding nucleic acid-nucleic acid-duplex. Typically there willbe an increase in T_(m) of about 1° C. per base pair at 100 mM NaCldepending on the sequence (Egholm et al. (1993), Nature, 365, 566-568).

[0208] In contrast to DNA-DNA-duplex formation, no salt is necessary tofacilitate and stabilise the formation of a peptide nucleic acid-DNA ora peptide nucleic acid-RNA duplex. The T_(m) of the peptide nucleicacid-DNA-duplex changes only little with increasing ionic strength.Typically for a 15-mer, the T_(m) will drop only 5° C. when the saltconcentration is raised from 10 mM NaCl to 1 M NaCl. At low ionicstrength (e.g. 10 mM phosphate buffer with no salt added), hybridisationof a peptide nucleic acid to a target sequence is possible underconditions where no stable DNA-DNA-duplex formation occurs. Furthermore,target sites that normally are inaccessible can be made more readilyaccessible for hybridisation with peptide nucleic acid probes at lowsalt concentration as the secondary and tertiary structure of nucleicacids are destabilised under such conditions. Using peptide nucleic acidprobes, a separate destabilising step or use of destabilising probes maynot be necessary to perform.

[0209] The rRNAs are essential for proper function of the ribosomes andthus the synthesis of proteins. The genes encoding the rRNAs are ineubacteria located in an operon in which the small subunit RNA gene, the16S rRNA gene, is located nearest the 5′ end of the operon, the gene forthe large subunit RNA, the 23S rRNA gene, is located distal to the 16SrRNA gene and the 5S rRNA gene is located nearest the 3′ end of theoperon. The three genes are separated by spacer regions in which tRNAgenes may be found, however, there are none in M. tuberculosis. Theprimary transcript of the eubacterial rRNA operon is cleaved byRNaselll. This cleavage results in separation of the 16S, the 23S andthe 5S rRNA into precursor rRNA molecules (pre-rRNA molecules) whichbesides the rRNA species also contain leader and tail sequences. Theprimary RNase IlIl cleavage is normally a rapid process, whereas thesubsequent maturation is substantially slower. Precursor rRNA istypically more abundant than even strongly expressed mRNA species. Thus,for certain applications, precursor rRNA may be an attractive diagnostictarget. In order to specifically detect precursor rRNA, a target probeshould be directed against sequences comprising at least part of theleader or tail sequences. A target probe may further be directed againstsequences of which both part of the leader/tail and mature rRNAsequences are constituents.

[0210] Usually, patients having contracted a mycobacterial infection aretreated with medicaments until no mycobacteria can be found in thesputum. Except for culturing, the presently available methods do notallow for clear distinguishing between living and dead mycobacteria.This means that a patient may often be treated with medicaments for alonger period of time than actually necessary. A way of determining theprogress of treatment would be a very valuable tool in the fight oftuberculosis and other mycobacterial diseases.

[0211] As transcription and maturation of rRNA is a measure ofviability, detection of precursor rRNA is a suitable and direct measureof viability of the bacteria. Furthermore, precursor rRNA may be usedfor identification of antibiotic drugs which reduce or inhibit rRNAtranscription. One such example is rifampicin. A transcriptionalinhibitor will in susceptible bacteria eliminate new synthesis of rRNAand thus the pool of precursor rRNA will be depleted. However, inresistant cells, primary transcripts as well as precursor rRNAs willcontinue to be produced.

[0212] Although it is preferred to use peptide nucleic acid probestargeting specific sequences of rRNA, it will readily be understood thatpeptide nucleic acid probes complementary to rRNA targeting probes willbe useful for the detection of the genes coding for said sequencespecific rRNA (rDNA), and peptide nucleic acid probes for the detectingrDNA is hence contemplated by the present invention. Although it ispreferred to choose the sequence of the probe so as to enable the probeto hybridise to its target sequence in antiparallel orientation, it isto be understood that probes capable of hybridising in parallelorientation can be constructed from the same information. The presentinvention is intended to cover both types of probes.

[0213] In the broadest aspect, the present invention relates to peptidenucleic acid probes for detecting a target sequence of one or moremycobacteria optionally present in a test sample, said probe beingcapable of hybridising to a target sequence of mycobacterial rDNA,precursor rRNA or rRNA.

[0214] The probes of the invention may suitably be directed to rDNA,precursor rRNA, or to 23S, 16S or 5S rRNA.

[0215] The target sequences, to which the peptide nucleic acid probe(s)are capable of hybridising to, are obtainable by

[0216] (a) comparing the nucleobase sequences of said mycobacterial rRNAor rDNA of one or more mycobacteria to be detected with thecorresponding nucleobase sequence of organism(s), in particular othermycobacteria, in particular other mycobacteria, from which said one ormore mycobacteria are to be distinguished,

[0217] (b) selecting a target sequence of said rRNA or rDNA whichincludes at least one nucleobase differing from the correspondingnucleobase of the organism(s), in particular other mycobacteria, fromwhich said one or more mycobacteria are to be distinguished, and

[0218] (c) determining the capability of said probe to hybridise to theselected target sequence to form detectable hybrids.

[0219] Peptide nucleic acid probes are obtainable by

[0220] (a) comparing the nucleobase sequences of said mycobacterial rRNAor rDNA of one or more mycobacteria to be detected with thecorresponding nucleobase sequence of organism(s), in particular othermycobacteria, in particular other mycobacteria, from which said one ormore mycobacteria are to be distinguished,

[0221] (b) selecting a target sequence of said rRNA or rDNA whichincludes at least one nucleobase differing from the correspondingnucleobase of the organism(s), in particular other mycobacteria, fromwhich said one or more mycobacteria are to be distinguished,

[0222] (c) synthesising said probe, and

[0223] (4) determining the capability of said probe to hybridise to theselected target sequence to form detectable hybrids.

[0224] The probes are in particular suitable for detecting a targetsequence of one or more mycobacteria of the Mycobacterium tuberculosisComplex (MTC) or for detecting a target sequence of one or moremycobacteria other than mycobacteria of the Mycobacterium tuberculosisComplex (MOTT) optionally present in a sample, which probe comprisesfrom 6 to 30 polymerised peptide nucleic acid moieties, said probe beingcapable of hybridising to a target sequence of mycobacterial rDNA,precursor rRNA or 23S, 16S or 5S rRNA forming detectable hybrids. Suchprobes may comprise peptide nucleic acid moieties ot formula (I)

[0225] wherein each X and Y independently designate O or S,

[0226] each Z independently designates O, S, NR¹, or C(R¹)₂, whereineach R¹ independently designate H, C₁₋₆ alkyl, C₁₋₆ alkenyl, C₁₋₆alkynyl,

[0227] each R², R³ and R⁴ designate independently H, the side chain of anaturally occurring amino acid, the side chain of a non-naturallyoccurring amino acid, C₁₋₄ alkyl, C₁₋₄ alkenyl or C₁₋₄ alkynyl, or afunctional group, each Q independently designates a naturally occurringnucleobase, a non-naturally occurring nucleobase, an intercalator, anucleobase-binding group, a label or H, with the proviso indicatedabove.

[0228] The probes may suitably be used for detecting a species specificmycobacterial target sequence, or target sequences of a group ofmycobacteria like MTC, MOTT, MAC or MAIS. The probes may further bedesigned so as to be capable of hybridising to one or more drugresistant mycobacteria, or, alternatively, to the wild-typecorresponding thereto. In the design of the probes, sequences betweendifferent mycobacteria (one or more) may be taken into account as maysequences from other related or non-related organisms (one or more).

[0229] As mentioned above, drug-resistance is an increasing threat tothe fight of mycobacterial infection. Monotherapy with macrolides suchas clarithromycin and azithromycin often leads to clinically significantdrug-resistance. Clarithromycin and azithromycin are important drugs inthe treatment of infections by especially M. avium. Comparison between23S rRNA sequences from isolates of M. avium and M. intracellulare withacquired resistance to clarithromycin and azithromycin and 23S rRNAsequences from non-resistant strains has revealed that the majority ofresistant strains have single-point mutations in the 23S rRNA inpositions corresponding to 2058 and 2059 in E. coli 23S rRNA. In the M.avium 23S rRNA sequence (GenBank accession number X74494), thecorresponding bases are in position 2568 and 2569, respectively, asshown in FIG. 6. Most susceptible strains have an A residue in one ofthese positions whereas the resistant strains have a C, G or T inposition 2058 (E. coli numbering corresponding to 2568 in M. avium withGenBank accession number X74494), or G or C in position 2059 (E. colinumbering corresponding to 2569 in M. avium with GenBank accessionnumber X74494).

[0230] Single-point mutations in rRNA apparently also account to somedegree for streptomycin resistance. Streptomycin, the first successfulantibiotic drug against tuberculosis, is an aminocyclitol glycoside thatperturbs protein synthesis at the ribosomal level. The genetic basis forstreptomycin resistance has not yet been completely solved. However.some streptomycin resistant strains of M. tuberculosis have single-pointmutations in 16S rRNA. These mutations are located in positionscorresponding to bases 501, 522, 523, 526, 912 and 913 in E. coli 16SrRNA which correspond to bases with numbers 452, 473, 474, 477, 865 and866, respectively, of M. tuberculosis 16S rRNA (GenBank accession numberX52917) as shown in FIG. 7. Most of these mutated nucleotides areinvolved in structural interactions within the 530 loop of 16S rRNAwhich is one of the most conserved regions in the entire 16S rRNA gene.

[0231] Mutations in an 81 bp region of the gene (rpoB) encoding the betasubunit of RNA polymerase are the cause of 96% of the cases ofrifampicin resistance in M. tuberculosis and M. Ieprae. rRNA precursormolecules have terminal domains (tails) which are removed during latesteps in precursor rRNA processing to yield the mature rRNA molecules.Transcriptional inhibitors such as rifampicin can deplete precursor rRNAin sensitive cells by inhibiting de novo precursor rRNA synthesis whileallowing maturation to proceed. Thus, precursor rRNA is depleted insusceptible mycobacterium cells while it remains produced in resistantmycobacterium cells when the cells are exposed to rifampicin duringculturing.

[0232] Peptide nucleic acid probes for detecting a target sequence ofone or more mycobacteria of the Mycobacterium tuberculosis Complex aredefined above. Peptide nucleic acid probes for detecting a targetsequence of one or more mycobacteria other than mycobacteria of theMycobacterium tuberculosis Complex are defined above. Peptide nucleicacid probes for detecting a target sequence of one or more drugresistant mycobacteria of the Mycobacterium tuberculosis complex or ofone or more drug resistant mycobacteria other than mycobacteria of theMycobacterium tuberculosis Complex are defined above.

[0233] In the present context and the claims, the term “naturallyoccurring nucleobases” includes the four main DNA bases (i.e. thymine(T), cytosine (C), adenine (A) and guanine (G)) as well as othernaturally occurring nucleobases (e.g. uracil (U) and hypoxanthine).

[0234] The term “non-naturally occurring nucleobases” comprises i.a.modified naturally occurring nucleobases. Such non-naturally occurringnucleobases may be modified by substitution by e.g. one or more C₁₋₈alkyl, C₁₋₈ alkenyl or C₁₋₈ alkynyl groups or labels. Examples ofnon-naturally occurring nucleobases are purine, 2,6-diamino purine,5-propynylcytosine (C propynyl), isocytosine (iso-C),5-methyl-isocytosine (iso^(Me)C) (see e.g. Tetrahedron Letters Vol 36,No 12, 2033-2036 (1995) or Tetrahedron Letters Vol 36, No 21, 3601-3604(1995)), 7-deazaadenine, 7-deazaguanine, N⁴-ethanocytosine,N⁶-ethano-2,6-diaminopurine, 5-(C₃₋₆)-alkenyluracil,5-(C₃₋₆)-alkynylcytosine, 5-fluorouracil and pseudocytosine.

[0235] Examples of Useful Intercalators are e.g. Acridin, Antraquinone,Psoralen and Pyrene.

[0236] Examples of useful nucleobase-binding groups are e.g. groupscontaining cyclic or heterocyclic rings. Non-limiting examples are3-nitro pyrrofe and 5-nitro indole.

[0237] It is to be understood that alkyl, alkenyl and alkynyl groups maybe branched or non-branched, cyclic or non-cyclic, and may further beinterrupted by one or more heteroatoms, or may be unsubtituted orsubstituted by or may contain one or more functional groups.Non-limiting examples of such functional groups are acetyl groups, acylgroups, amino groups, carbamido groups, carbamoyl groups, carbamylgroups, carbonyl groups, carboxy groups, cyano groups, dithio groups,formyl groups, guanidino groups, halogens, hydrazino groups, hydrazogroups, hydroxamino groups, hydroxy groups, keto groups, mercaptogroups, nitro groups, phospho groups, phosphono groups, phospho estergroups, sulfo groups, thiocyanato groups, cyclic, aromatic andheterocyclic groups.

[0238] C₁₋₄ groups contain from 1 to 4 carbon atoms, C₁₋₆ groups containfrom 1 to 6 carbon atoms, and C₁₋₁₅ groups contain from 1 to 15 carbonatoms, not including optional substituents, heteroatoms and/orfunctional groups. Non-limiting examples of such groups are —CH₃, —CF₃,—CH₂—, —CH₂CH₃, —CH₂CH₂—, —CH(CH₃)₂, —OCH₃, —OCH₂—, —OCH₂CH₃, —OCH₂CH₂—,—OCH(CH₃)₂, —OC(O)CH₃, —OC(O)CH₂—, —C(O)H, —C(O)—, —C(O)CH₃, —C(O)OH,—C(O)O—, —CH₂NH₂, —CH₂NH—, —CH₂OCH₃, —CH₂OCH₂—, —CH₂OC(O)OH,—CH₂OC(O)O—, —CH₂C(O)CH₃, —CH₂C(O)CH₂—, —C(O)NH₂, —CH═CH₂, —CH═CH—,—CH═CHCH₂C(O)OH, —CH═CHCH₂C(O)O—, —C≡CH, —C≡C—, —CH₂C≡CH, —CH₂C≡C—,—CH₂C≡CCH₃, —OCH₂C≡CH, —OCH₂C≡C—, —OCH₂C≡CCH₃, —NHCH₂C(O)—,—NHCH₂CH₂C(O)—, —NH(CH₂CH₂O)₂CH₂C(O)—, andHO(O)CCH₂C(O)(NH—(CH₂CH₂O))₂—, phenyl, benzyl, naphthyl, oxazolyl,pyridinyl, thiadiazolyl, triazolyl, and thienyl.

[0239] Within the present context, the expression “naturally occurringamino acid” is intended to comprise D- and L-forms of amino acidscommonly found in nature, e.g. D- and L-forms of Ala (alanine), Arg(arginine), Asn (aspargine), Asp (aspartic acid), Cys (cysteine), Gln(glutamine), Glu (glutamic acid), His (histidine), Ile (isoleucine), Leu(leucine), Lys (lysine), Met (methionine), Phe (phenylalanine), Pro(proline), Ser (serine), Thr (threonine), Trp (tryptophan), Tyr(tyrosine) and Val (valine).

[0240] In the present context, the expression “non-naturally occurringamino acid” is intended to comprise D- and L-forms of amino acids otherthan those commonly found in nature as well as modified naturallyoccurring amino acids. Examples of useful non-naturally occurring aminoacids are D- and L-forms of β-Ala (β-alanine) Cha (cyclohexylalanine),Cit (citrulline), Hci (homocitrulline), HomoCys (homocystein), Hse(homoserine), NIe (norleucine), Nva (norvaline), Orn (ornithine), Sar(sarcosine) and Thi (thienylalanine).

[0241] In the present context, the term “sample” is intended to coverall types of samples suitable for the purpose of the invention. Examplesof such samples are sputum, laryngeal swabs, gastric lavage, bronchialwashings, biopsies, aspirates, expectorates, body fluids (spinal,pleural, pericardial, synovial, blood, pus, bone marrow), urine, tissuesections as well as food samples, soil, air and water samples. Analysisof samples originating from the before-mentioned samples (e.g. culturesand treated samples) are also within the scope of the invention.

[0242] In the present context, the term “hybrids” is intended to includecomplexes between a probe and a nucleic acid to be determined. Suchhybrids may be made up of two or more strands.

[0243] The strength of the binding between the probe and the targetnucleic acid sequence may be influenced by the ligand Q. When Qdesignates a nucleobase, Hoogsteen and/or Watson-Crick base pairingassist(s) in the formation of hybrids between a nucleic acid sequence tobe detected and a probe. It is contemplated that one or more of theligands may be a group which contribute little or none to the binding ofthe nucleic acid such as hydrogen. It is contemplated that suitableprobes to be used comprise less than 25% by weight of peptide nucleicacid moieties, wherein Q designates such groups. One or more of theligands Q may be groups that stabilise nucleobase stacking such asintercalators or nucleobase-binding groups.

[0244] In the above-indicated probes, one or more of the Q-groups maydesignate a label. Examples of suitable labels are given below. Moietieswherein Q denotes a label may preferably be located in one or both ofthe terminating moieties of the probe. Moieties wherein Q denotes alabel may, however, also be located internally.

[0245] The peptide nucleic acid probes may comprise moieties, whereinall X groups are O (polyamides) or wherein all X groups are S(polythioamides). It is to be understood that the probes may alsocomprise mixed moieties (comprising both amide and thioamide moieties).

[0246] In another aspect, the present invention relates to peptidenucleic acid probes of formula (II), (III) and (IV) as well as mixturesof such probes defined above.

[0247] In a preferred embodiment, the peptide nucleic acid probes ormixtures thereof according to the invention are of formulas (I)-(IV) asdefined above with Z being NH, NCH₃ or O, each R², R³ and R⁴independently being H or the side chain of a naturally occurring aminoacid, the side chain of a non-naturally occurring amino acid, or C₁₋₄alkyl, and each Q being a naturally occurring nucleobase or anon-naturally occurring nucleobase with the provisos defined above.

[0248] Peptide nucleic acid probes or mixtures of such probes accordingto the invention are preferably those of formula (I)-(IV) as definedabove with Z being NH or O, and R² being H or the side chain of Ala,Asp, Cys, Glu, His, HomoCys, Lys, Orn, Ser or Thr, and Q being anucleobase selected from thymine, adenine, cytosine, guanine, uracil,iso-C, and 2,6-diaminopurine with the provisos defined above.

[0249] Peptide nucleic acid probes or mixtures thereof, which are ofmajor interest for detecting mycobacteria of the MTC group or one ormore mycobacteria other than mycobact3 eria of the MTC group, are probesof formula (V) as defined above, wherein R⁴ is H or the side chain ofAla, Asp, Cys, Glu, His, HomoCys, Lys, Orn, Ser or Thr, Q is as definedabove and with the provisos indicated above.

[0250] The peptide nucleic acid probe comprises polymerised moieties asdefined above and in the claims. From the formula, it is to beunderstood that the probe may comprise polymerised moieties whichstructure may be mutually different or identical. In some cases, it maybe advantageous that at least one moiety of the probe, preferably one(or both) of the moieties terminating the probe, are of a differentstructure. Such terminating moieties may suitably be a moiety of formula(VI)

[0251] where Q is as defined above. Such moiety may suitably beconnected to a peptide nucleic acid moiety through an amide bond.

[0252] The peptide nucleic acid probe according to the inventioncomprises from 6 to 30 polymerised moieties of formulas (I) to (V), and,in addition, optionally one or two terminating moieties of formula (VI)as defined above. The preferred length of the probe will depend on thesample material and whether labelled probes are used. It is contemplatedthat especially interesting probes comprise from 10 to 30 polymerisedmoieties of formulas (I) to (V), and, in addition, optionally one or twoterminating moieties of formula (VI) as defined above. Probes of theinvention may suitably comprise from 12 to 25 polymerised moieties offormulas (I) to (V), more suitably from 14 to 22 polymerised moieties offormulas (I) to (V), most suitably from 15 to 20 polymerised moieties offormulas (I) to (V), and, in addition, optionally one or two terminatingmoieties of formula (VI).

[0253] As mentioned above, the polymerised moieties of the probes may bemutually different or identical. In some embodiments, the polymerisedmoieties of formulas (V) constitute at least 75% by weight (calculatedby excluding labels and linkers), preferably at least 80% by weight andmost preferably at least 90% by weight of the probe.

[0254] The ends on the moieties terminating the probe may be substitutedby suitable substituents which in the following will be named “linkers”.A terminating end may suitably be substituted by from 1 to 5 linkers,more suitably from 1 to 3 linkers. Such linkers may suitably be selectedamong C₁₋₁₅ alkyl, C₁₋₁₅ alkenyl and C₁₋₁₅ alkynyl groups as definedabove. The linkers may be substituted or unsubstituted, branched ornon-branched, or be interrupted by heteroatoms, or be substituted orcontain functional groups as described above. This may depend on thechemical nature of the terminating moiety (i.e. whether the moiety isterminated by a carbon, oxygen or nitrogen atom). A terminating end or alinker on a terminating end may further be substituted by one or morelabels, which labels may be incorporated end to end, i.e. so as to forma non-branched labelled end, or may be incorporated so as to form abranched labelled end (“zipper”). The linkers may be attached directlyto a terminating end, may be attached to a label or between labels on aterminating end, or be attached to a terminating end before a label isattached to a terminating end. It should be understood that twoterminating ends may carry different or identical substituents, linkersand/or labels. It should further be understood that the term “a label”is intended to comprise one or more labels as the term “linkers” is tocomprise one or more linkers. For certain applications, it may beadvantageous that one or more linkers are incorporated between thepeptide nucleic acid moieties. Such applications may in particular bethose based on triplex formation.

[0255] Examples of suitable linkers are —NH(CH₂CH₂O)_(n)CH₂C(O)—,—NH(CHOH)_(n)C(O)—, —(O)C(CH₂OCH₂)_(n)C(O)— and —NH(CH₂)_(n)C(O)—,NH₂(CH₂CH₂O)_(n)CH₂C(O)—, NH₂(CHOH)_(n)C(O)—, HO(O)C(CH₂OCH₂)_(n)C(O)—,NH₂(CH₂)_(n)C(O)—, —NH(CH₂CH₂O)_(n)CH₂C(O)OH, —NH(CHOH)_(n)C(O)OH,—(O)C(CH₂OCH₂)_(n)C(O)OH and —NH(CH₂)_(n)C(O)OH, wherein n is 0 or aninteger from 1 to 8, preferably from 1 to 3. Examples of veryinteresting linkers are —NHCH₂C(O)—, —NHCH₂CH₂C(O)—,—NH(CH₂CH₂O)₂CH₂C(O)—, and HO(O)CCH₂CH₂C(O)(NH(CH₂CH₂O)₂CH₂C(O))₂—.

[0256] In the present context, the term “label” refers to a substituentwhich is useful for detection or visualisation. Suitable labels comprisefluorophores, biotin, dinitro benzoic acid, digoxigenin, radioisotopelabels, peptide or enzyme labels, chemiluminiscence labels, fluorescentparticles, hapten, antigen or antibody labels.

[0257] The expression “peptide label” is intended to mean a labelcomprising from 1 to 20 naturally occurring or non-naturally occurringamino acids, preferably from 1 to 10 naturally occurring ornon-naturally occurring amino acids, more preferably from 1 to 8naturally occurring or nonnaturally occurring amino acids, mostpreferably from 1 to 4 naturally occurring or non-naturally occurringamino acids, linked together end to end in a non-branched or branched(“zipper”) fashion. Such peptide label may be composed of amino acidswhich are mutually identical or different. In a preferred embodiment,such a non-branched or branched end comprises one or more, preferablyfrom 1 to 8 labels, more preferably from 1 to 4, most preferably 1 or 2,further labels other than a peptide label. Such further labels maysuitably terminate a non-branched end or a branched end. One or morelinkers may suitably be attached to the terminating end before a peptidelabel and/or a further label is attached. Such linker units may also beattached between a peptide label and a further label. Furthermore, suchpeptide labels may be incorporated between the peptide nucleic acidmoieties.

[0258] The probe as such may also comprise one or more labels such asfrom 1 to 8, preferably from 1 to 4, most preferably 1 or 2, labelsand/or one or more linker units, which may be attached internally, i.e.to the backbone of the probe. The linker units and labels may. mutuallybe attached as described above.

[0259] Examples of particular interesting labels are biotin, fluoresceinlabels, e.g. 5-(and 6)-carboxyfluorescein, 5- or 6-carboxyfluorescein,6-(fluorescein)-5-(and 6)-carboxamido hexanoic acid and fluoresceinisothiocyanate, peptide labels consisting of from 1 to 20 naturallyoccurring amino acids or non-naturally occurring amino acids, enzymelabels such as peroxidases like horse radish peroxidase (HRP), alkalinephosphatase, and soya bean peroxidase, dinitro benzoic acid, rhodamine,tetramethylrhodamine, cyanine dyes such as Cy2, Cy3 and Cy5, coumarin,R-phycoerythrin (RPE), allophycoerythrin, Texas Red, Princeton Red, andOregon Green as well as conjugates of R-phycoerythrin and, e.g. Cy5 orTexas Red.

[0260] Examples of preferred labels are biotin, fluorescent labels,peptide labels, enzyme labels and dinitro benzoic acid. Peptide labelsmay preferably be composed of from 1 to 10, more preferably of from 1 to8, most preferably of from 1 to 4, naturally occurring or non-naturallyoccurring amino acids. It may be particularly advantageous toincorporate one or more other labels as well as a peptide label such asfrom 1 to 8 or from 1 to 4 other labels, preferably 1 or 2 other labels.

[0261] Suitable peptide labels may preferably be composed of cysteine,glycine, lysine or ornithine.

[0262] In a further embodiment, the Q substituent as defined above maybe labelled. Suitable labels are as defined above. Between Q and such alabel, a linker as defined above may be incorporated. It is preferredthat such labelled ligands Q are selected from thymine and uracillabelled in the 5-position and 7-deazaguanine and 7-deazaadeninelabelled in the 7-position.

[0263] A mixture of peptide nucleic acid probes is also part of thepresent invention. Such mixture may comprise more than one probe capableof hybridising to 23S rRNA, and/or more than one probe capable ofhybridising to 16S rRNA, and/or or more than one probe capable ofhybridising to 5S rRNA. A mixture of probes may further compriseprobe(s) directed to precursor rRNA and/or rDNA. The mixture may alsocomprise peptide nucleic acids for detecting more than one mycobacteriain the same assay.

[0264] In a preferred embodiment, the nucleobase sequence of the peptidenucleic acid probe is selected so as to be substantially complementaryto the nucleobase sequence of the target sequence in question. In anespecially preferred embodiment, the nucleobase sequence of the peptidenucleic acid probe is selected so as to be complementary to thenucleobase sequence of the target sequence in question. By“complementary” is meant that the nucleobases are selected so as toenable perfect match between the nucleobases of the probe and thenucleobases of the target, i.e. A to T or G to C. By substantiallycomplementary is meant that the peptide nucleic acid probe is capable ofhybridising to the target sequence, however, the probe does notnecessarily have to be perfectly complementary to the target. Forexample, probes comprising one or more bases not complementary to thetarget sequence and nontarget sequences, especially base(s) located atthe end of the probe, where the effect on the stability of probe-targetnucleic acid hybrids is low. Another example is probes comprising othernaturally occurring bases. Thus provided that the probe is capable ofhybridising to the target sequence, the nucleobase difference(s) betweentarget sequences and non-target sequences ensures that the stability ofprobe-non-target nucleic acid hybrids are lower than the stability ofprobe-target nucleic acid hybrids and therefore make such substantiallycomplementary probes applicable for detection of mycobacteria.

[0265] The probes may be synthesised according to the proceduresdescribed in “PNA Information Package” obtained from MilliporeCorporation (Bedford, Mass., USA), or may be synthesised on an ExpediteNucleic Acid Synthesis System (PerSeptive BioSystems, USA).

[0266] If using the Fmoc strategy for elongation of the probe withlinkers or amino acids, it is possible to retain side chain amino groupsprotected with acid sensitive protection groups such as the Boc or Mttgroup. This method allows introduction of a linker containing severalBoc protected amino groups which can all be cleaved and labelled in thesame synthesis cycle.

[0267] One way of labelling a probe is to use a fluorescent label, suchas 5-(and 6)-carboxyfluorescein, 5- or 6-carboxyfluorescein, or6-(fluorescein)-5-(and 6)-carboxamido hexanoic acid. The acid group isactivated with HATU(O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate) or HBTU(2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate) and reacted with the N-terminal amino group of thepeptide nucleic acid. The same technique can be applied to otherlabelling groups containing an acid function.

[0268] Alternatively, the succinimidyl ester of the above-mentionedlabels may suitably be used or fluorescein isothiocyanate may be useddirectly.

[0269] After synthesis, probes can be cleaved from the resin usingstandard procedures as described by Millipore Corporation or PerSeptiveBioSystems. The probes are subsequently purified and analysed usingreversed-phase HPLC techniques at 50° C. and were characterised bymatrix-assisted laser desorption/ionisation time of flight massspectrometry (MALDI-TOFMS), plasma desorption mass spectrometry (PDMS),electron spray mass spectrometry (ESMS), or fast atom bombardment(FAB-MS).

[0270] Generally, probes such as probes comprising polymerised moietiesof formula (IV) and (V) may also be prepared as described in, e g.,Angewandte Chemie, International Edition in English 35, 1939-1942 (1996)and Bioorganic & Medical Chemistry Letters, Vol 4, No 8, 1077-1080(1994). Chemical properties of some probes are described in, e.g.,Nature, 365, 566-568 (1993).

[0271] The method as claimed can be used for the detection of a targetsequence of one or more mycobacteria optionally present in a sample. Themethod and the probes provide a valuable tool for analysing samples forthe presence of such target sequences, hence providing information forestablishing a diagnosis.

[0272] In the assay method according to the invention, the sample to beanalysed for the presence of mycobacteria is brought into contact withone or more probes or a mixture of such probes according to theinvention under conditions by which hybridisation between the probe(s)and any sample rRNA or rDNA originating from mycobacteria can occur, andthe formed hybrids, if any, are observed or measured, and theobservation or measurement is related to the presence of a targetsequence of one or more mycobacteria. The observation or measurement maybe accomplished visually or by means of instrumentation.

[0273] Prior to contact with probe(s) according to the invention, thesamples may undergo various types of sample processing which includepurification, decontamination andlor concentration. The sample maysuitably be decontaminated by treatment with sodium hypochlorite andsubsequently centrifuged for concentration of the mycobacteria. Samplese.g. sputum samples may be treated with a mucolytic agent such asN-Acetyl-L-cystein which reduces the viscosity of the sample as well asbe treated with sodium hydroxide which decontaminates the sample, andsubsequently centrifuged. Other well-known decontamination andconcentration procedures include the Zephiran-trisodium phosphatemethod, Petroffs sodium hydroxide method, the oxalic acid method as wellas the cetylpyridinium chloride-sodium chloride method. Samples may alsobe purified and concentrated by applying sample preparation methods suchas filtration, immunocapture, two-phase separation either alone or incombination. The sample preparation methods may also be used togetherwith the centrifugation and decontamination methods mentioned above.

[0274] Samples may, either directly or after having undergone one ormore processing steps, be analysed in primarily two major types ofassays, in situ-based assays and in vitro-based assays. In this context,in situ-based assays are to be understood as assays, in which the targetnucleic acids remain within the bacterial cell during the hybridisationprocess. Examples are in situ hybridisation (ISH) assays on smears andbiopsies as well as hybridisation to whole cells which may be insuspension and which subsequently may be analysed by e.g. flow cytometryoptionally after capture of the bacteria onto particles (with same ordifferent type and size), or by image analysis after spreading of thebacteria onto a solid medium, filter membrane or another substantiallyplanar surface.

[0275] In vitro-based assays are to be understood as assays, in whichthe target nucleic acids are released from the bacterial cell beforehybridisation. Examples of such assays are microtiter plate-basedassays. Many other assay types, in which the released target nucleicacids by some means are captured onto a solid phase and subsequentlyanalysed via a detector probe, are feasible and within the scope of thepresent invention. Even further, in vitro-based assays include assays,in which the target nucleic acids are not captured onto a solid phase,but in which the hybridisation and signal generation take place entirelyin solution.

[0276] Samples for in situ-based assays may suitably be applied andoptionally be immobilised to a support. Techniques for applying of asample onto a solid support depend on the type of sample in question andinclude smearing and cytocentrifugation for liquid or liquified samplesand sectioning of tissues for biopsy materials. The solid support maytake a wide variety of forms well-known in the art, such as a microscopeslide, a filter membrane, a polymer membrane or a plate of variousmaterials.

[0277] In the case of in vitro-based assays, the target nucleic acid maybe released from the mycobacterial cells in various ways. Most methodsfor releasing the nucleic acids cause bursting of the cell wall (lysis)followed by extraction of the nucleic acids into a buffered solution. Asmycobacteria have complex cell walls containing covalently associatedpeptidoglycans, arabinogalactans and in particular mycolic acids, theycannot easily be disrupted by standard methods used for the rapid lysisof other bacteria. Possible methods which are known to give successfullysis of the mycobacterial cell wall include methods which involvetreatment with organic solvents, treatment with strong chaotropicreagents such as high concentrations of guanidine thiocyanate, enzymetreatment, bead beating, heat treatment, sonication and/or applicationof a French press.

[0278] Samples to be analysed by in situ assays may be fixed prior tohybridisation. The person skilled in the art will readily recognise thatthe appropriate procedure will depend on the type of sample to beexamined. Fixation and/or immobilisation should preferably preserve themorphological integrity of the cellular matrix and of the nucleic acids.Examples of methods for fixation are flame fixation, heat fixation,chemical fixation and freezing. Flame fixation may be accomplished bypassing the slide through the blue cone of a Bunsen burner 3 or 4 times;heat fixation may be accomplished by heating the sample to 80° C. for 2hours; chemical fixation may be accomplished by immersion of the samplein a fixative (e.g. formamide, methanol or ethanol). Freezing isparticularly relevant for biopsies and tissue sections and is usuallycarried out in liquid nitrogen.

[0279] In one in situ hybridisation assay embodiment, the sample to beanalysed is smeared onto a substantially planar solid support which maybe a microscope slide, a filter membrane, a polymer membrane or anothertype of solid support with a planar surface. The preferred solid supportis a microscope slide. After the smear has been prepared, it mayoptionally undergo further pre-treatment like treatment withbactericidal agents or additional fixation by immersion in e.g. ethanol.The sample may also be pre-treated with enzyme(s) which as primaryfunction permeabilise the cells and/or reduce the viscosity of thesample. It may further be advantageous to perform a pre-hybridisationstep in order to block sites which might otherwise give raise tonon-specific binding. For this purpose, blocking agents like skim milk,and nontarget probes may suitably be used. The components of thepre-hybridisation mixture should be selected so as to obtain aneffective saturation of sites in the sample that might otherwise bindthe probe non-specifically. The pre-hybridisation buffer may suitablycomprise an appropriate buffer, blocking agent(s), and detergents.

[0280] During the in situ hybridisation, one or more probes according tothe present invention are brought into contact with any target rRNA orrDNA inside the cells in a hybridisation solution under suitablestringency conditions. The concentration of the applied probe may varydepending on the chemical nature of the probe and the conditions underwhich hybridisation is carried out. Typically, a probe concentrationbetween 1 nM and 1 μM is suitable. The hybridisation solution maycomprise a denaturing agent which allows hybridisation to take place ata lower temperature than would be the case without the agent. Thedenaturing agent should be present in an amount effective to increasethe ratio between specific binding and non-specific binding. Theeffective amount of denaturing agent depends on the type used and on theprobe or combination of probes. Examples of denaturing agents areformamide, ethylene glycol and glycerol, and these may preferably beused in a concentration above 10% and less than 70%. The preferreddenaturing agent is formamide which is used more preferably inconcentrations from 20% to 60%, most preferably from 30% to 50%. Itshould be noted that in several instances it may not be necessary oradvantageous to include a denaturing agent.

[0281] Prior to hybridisation or during hybridisation, a mixture ofrandom probes (probes with random, non-selected sequences of optionallydifferent length) may be added in excess to reduce non-specific binding.Also, one or more non-sense probes (probes with a defined nucleobasesequence and length differing from the nucleobase sequence of the targetsequence) may be added in excess in order to reduce non-specificbinding. Also, non-specific binding of detectable probes to one or morenon-target nucleic acid sequences can be suppressed by addition of oneor more unlabelled or independently detectable probes, which probes havea sequence that is complementary to the non-target sequence(s). It isparticularly advantageous to add such blocking probes when thenon-target sequence differs from the target sequence by only onemismatch.

[0282] It may be advantageous to include inert polymers which arebelieved to increase the effective concentration of the probe(s) in thehybridisation solution. One such macromolecule is dextran sulphate whichmay be used in concentrations of from 2.5% to 15%. The preferredconcentration range is from 8% to 12% in the case of dextran sulphate.Other useful macromolecules are polyvinylpyrrolidone and ficoll, whichtypically are used at lower concentrations, e.g. 0.2%. It may further beadvantageous to add one or more detergents which may reduce the degreeof non-specific binding of the peptide nucleic acid probes. Examples ofuseful detergents are sodium dodecyl sulphate, Tween 20® or TritonX-100®. Detergents are usually used in concentrations between 0.05% and1.0%, preferably between 0.05% and 0.25%. The hybridisation solution mayfurthermore contain salt. Although it is not necessary to include saltin order to obtain proper hybridisation, it may be advantageous toinclude salt in concentrations from 2 to 500 mM, or suitably from 5 to100 mM.

[0283] During hybridisation, other important parameters arehybridisation temperature, concentration of the probe and hybridisationtime. The person skilled in the art will readily recognise that optimalconditions must be determined for each of the above-mentioned parametersaccording to the specific situation, e.g. choice of probe(s) and typeand concentration of the components of the hybridisation buffer, inparticular the concentration of denaturing agent. Presence of volumeexcluders may also have an effect.

[0284] Following hybridisation, the sample is washed to remove anyunbound and any non-specifically bound probe, and consequently,appropriate stringency conditions should be used. By stringency is meantthe degree to which the reaction conditions favour the dissociation ofthe formed hybrids. The stringency may be increased typically byincreasing the washing temperature and/or washing time. Typically,washing times from 5 to 40 minutes may be sufficient. Two or morewashing periods of shorter time may also give good results. A range ofbuffers may be used, including biological buffers, phosphate buffers andstandard citrate buffers. The demand for low salt concentration in thebuffers is not as pertinent as for DNA probe assays due to thedifference response to salt concentration. In some cases, it isadvantageous to increase the pH of the washing buffer as it may give anincreased signal-to noise ratio (see WO 97/18325). This is conceivablydue to a significant reduction of the non-specific binding. Thus, it maybe advantageous to use a washing solution with a pH value form 8 to10.5, preferably from 9 to 10.

[0285] Visualisation of bound probe(s) must be carried out with dueregard to the type of label chosen. There are a wide range of usefulprobe labels, in particular various fluorescent labels such asfluorescein, rhodamine and derivatives thereof. Furthermore, labels likeenzymes (e.g. peroxidases and phosphatases) and haptens (e.g. biotin,digoxigenin, dinitro benzoic acid) may suitably be applied. In the caseof fluorescent labels, the hybrids formed may be visualised using amicroscope with a magnification of at least×250, preferably×1000. Thevisualisation may further be carried out using a CCD (charge coupleddevice) camera optionally controlled by a computer. When haptens areused as labels, the hybrids may be detected using an antibody conjugatedwith an enzyme. In these cases, the detection step may be based oncolorimetry, fluorescence or luminescence.

[0286] The probes may alternatively be labelled with fluorescentparticles having the fluorescent label embedded in the particles (e.g.Estapor K coulored microspheres), located on the surface of theparticles and/or coupled to the surfaces of the particles. As theparticles have to come into contact with the target nucleic acids withinthe cells, the use of fluorescent particles may necessitate pretreatmentof the bacteria. Relatively small particles e.g. about 20 nm maysuitable be used.

[0287] In another in situ hybridisation embodiment, frozen tissue orbiopsy samples are cut into thin sections and transferred to asubstantially planar surface, preferably microscope slides. Prior tohybridisation, the tissue or biopsy may be treated with a fixative,preferably a precipitating fixative such as acetone, or the sample isincubated in a solution of buffered formaldehyde. Alternatively, thebiopsy or tissue section can be transferred to a fixative such asbuffered formaldehyde for 12 to 24 hours and following fixation, thetissue may be embedded in paraffin forming a block from which thinsections can be cut. Prior to hybridisation, the tissue section isdewaxed and rehydrated using standard procedures. Permeabilisation (e.g.treatment with proteases, diluted acids, detergents, alcohol and/orheat) may in some cases be advantageous. The selected method forpermeabilisation depends on several factors, for instance on thefixative used, the extent of fixation, the type and size of sample, andon the applied probe. For these types of samples, sample processing,prehybridisation, hybridisation, washing and visualisation may becarried out using same or adjusted conditions as described above.

[0288] In a further embodiment of the in situ assays, the bacterialcells are kept in suspension during fixation, prehybridisation,hybridisation and washing are carried out under the same or similarconditions as described above. The preferred type of label for thisembodiment is fluorescent labels. This allows detection of hybridisedcells by flow cytometry, recording the intensity of fluorescence percell. Bacterial cells in suspension may further be coupled to particles,preferably with a size of from 20 nm to 10 μm. The particles may be madeof materials well-known in the art like latex, dextran, cellulose and/oragarose, and may optionally be paramagnetic or contain a fluorescentlabel. Normally, bacterial cells are coupled to particles usingantibodies against the target bacteria, but other means like molecularimprinting may also be used. Coupling of the bacterial cells toparticles may be advantageous in sample handling and/or duringdetection.

[0289] In the embodiments of in situ hybridisation described above, theprobes according to the invention are used for detecting a targetsequence of one or more mycobacteria. In a preferred embodiment, theprobes are suitable for detecting a target sequence of mycobacteria ofthe Mycobacterium tuberculosis Complex (MTC), mycobacteria other thanthe Mycobacterium tuberculosis Complex (MOTT), or mycobacteria of theMycobacterium avium Complex (MAC). The probes are further suitable fordetecting simultaneously different target sequences originating from thesame mycobacteria.

[0290] Samples to be analysed using in vitro-based assays need toundergo a treatment by which the nucleic acids are released from thebacterial cells. Nucleic acids may be released using organic solvents,strong chaotropic reagents such as high concentrations of guanidinethiocyanate, enzymes, bead beating, heating, sonication and/orapplication of a French press. The obtained nucleic acids may undergoadditional purification prior to hybridisation.

[0291] In one in vitro hybridisation embodiment, the sample comprisingthe target nucleic acid is added to a container comprising immobilisedcapture probe(s) and one or more probe(s) labelled to function asdetector probe(s). The hybridisation should be performed under suitablestringency conditions. The hybridisation solution may further comprise adenaturing agent, blocking probes, inert polymers, detergents and saltas described for the in situ-type assays. Likewise, the hybridisationtemperature, probe concentration and hybridisation time are importantparameters that need to be controlled according to the specificconditions of the assay, e.g. choice of peptide nucleic acid probe(s)and concentration of some of the ingredients of the hybridisationbuffer. If hybridisation of the target nucleic acid to the captureprobe(s) and detector probe(s), respectively, is performed in twoseparate steps, different parameters, in particular different stringencyconditions, may be used in these steps. The concentration of the captureprobe may be higher for in situ assays as hybridisation may becontrolled better and washing can be performed more efficiently.

[0292] The capture probes may be immobilised onto a solid support by anymeans, e.g. by a coupling reaction between a carboxylic acid on a linkerand an amino derivatised support. The capture probe may further becoupled onto the solid support by photochemical activation ofphotoreactive groups which have been attached absorptively to the solidsupport prior to photochemical activation. Such photoreactive groups aredescribed in the U.S. Pat. No. 5,316,784 A. The capture probes mayfurther be coupled to a hapten which allows an affinity basedimmobilisation to the solid support. One such example is coupling of abiotin to the probe(s) and immobilisation via binding to asteptavidin-coated surface.

[0293] The solid support may take a wide variety of forms well-known inthe art, such as a microtiter plate having one or more wells, a filtermembrane, a polymer membrane, a tube, a dip stick, a strip andparticles. Filter membranes may be made of cellulose, celluloseacetate,polyvinylidene fluoride or any other materials well-known in the art.The polymer membranes may be of polystyrene, nylon, polypropylene or anyother materials well known in the art. Particles may be paramagneticbeads, beads made of polystyrene, polypropylene, polyethylene, dextran,nylon, amyloses, celluloses, polyacrylamides and agarose. When the solidsupport has the form of a filter, a membrane, a strip or beads, it(they) may be incorporated into a single-use device.

[0294] The selection of the label of the detector probe(s) depend on thespecific assay format and possible instrumentation. When biotin labelledprobes are used, the hybrids may be detected using streptavidin or anantibody against the biotin label which antibody or streptavidin may beconjugated with an enzyme and the actual detection depend on the choiceof the specific enzyme, preferably a phosphatase or a peroxidase, andthe substrate for the selected enzyme The signal may in some cases beenhanced using commercially available amplification systems such as thecatalysed signal amplification system for biotinylated probes (CSA byDAKO). Various polymer-based enhancement systems may also be used. Anexample is a dextran polymer to which both a hapten specific antibodyand an enzyme is coupled. The detector probe(s) may further be labelledwith other haptens, e.g. digoxigenin, dinitro benzoic acid andfluorescein, in which case the hybrids may be detected using an antibodyagainst the hapten which antibody may be conjugated with an enzyme. Itis even possible to apply detector probe(s) which have enzymes coupleddirectly onto the probes. There are a wide range of possibilities forselection of enzyme substrates allowing for colourimetric (substratese.g. p-nitro-phenyl phosphate or tetra-methyl-benzidine), fluorogenic(substrates e.g. 4-methylumbilliferylphosphate) or chemiluminescent(substrates e.g. 1,2-dioxetanes) detection.

[0295] The detector probes may further be labelled with variousfluorescent labels, preferably fluorescein or rhodamine, in which casethe hybrids may be detected by measuring the fluorescence.

[0296] The detector probe(s) will typically be different from thecapture probe(s), thus ensuring dual species specificity. The dualspecificity will most often allow at least one of the probes to beshorter, e.g. a 10 mer probe.

[0297] Furthermore, the capture of purine rich sequences may be improvedby utilising bis-peptide nucleic acids as capture probes. Suchbis-peptide nucleic acids are described in WO 96/02558. The bis-peptidenucleic acids comprise a first peptide nucleic acid strand capable ofhybridising in parallel fashion to the target nucleic acid, and a secondpeptide nucleic acid strand capable of hybridising in antiparallelfashion to the purine rich sequence of the nucleic acid to be captured.The two peptide nucleic acid strands are connected by a linker and arein this way capable of forming a triplex structure with said purine richsequence. nucleic acid. The number of polymerised moieties of eachlinker-separated peptide nucleic acid may be as previously defined fornon-bis-peptide nucleic acids. However, due to the high stability of thetriplexes formed, bis-peptide nucleic acids with short first and secondstrands can be used making the design of a pyrimidine rich probe easier.

[0298] Instead of using a detector probe, capture probe: nucleic acidcomplexes may be detected using a detection system based on an antibodyreacting specifically with complexes formed between peptide nucleicacids and nucleic acids (such as described in WO 95/17430), in whichdetection system the primary antibody may comprise a label, or whichdetection system comprises a labelled secondary antibody, whichspecifically binds to the primary antibody. The specific detection againdepends on the selected substrate which may be of any type of thosementioned above.

[0299] Depending on the type of specific assay format, label anddetection principle various types of instrumentation may be usedincluding conventional microplate readers, luminometers and flowcytometers. Adaptation of adequate instrumentation may allow forautomatisation of the assay.

[0300] In an example of this embodiment, a capture probe of the presentinvention is coupled to a microtiter plate by a photochemical reactionbetween antraquinon-labelled capture probe and polystyrene of themicrowell. Target rRNA is added to the microwells and incubated understringent conditions. Unbound rRNA is removed by washing and themicrowell are incubated with a hapten-labelled detector probe understringent conditions. The visualisation is carried out using anenzyme-labelled antibody against the hapten, which after removal ofunbound antibody is detected using a chemiluminescence substrate.

[0301] In another example of this embodiment capture probes are coupledto latex particles, and hybridisation is carried out under suitableconditions in the presence of e.g. fluorescein labelled detectorprobe(s). After hybridisation and optionally washing, the hybrids aredetected by flow cytometry. A range of different beads (e.g. by size orcolours) may carry different capture probes for different targets, thusallowing a multiple detection system.

[0302] In a further embodiment of the in vitro assays format, thecapture probe, the target nucleic acid and the detector probe mayhybridise in solution, and subsequently the capture probe is attached toa solid phase. The solid phase, the hybridisation conditions and meansof detection may be selected according to the specific method asdescribed above.

[0303] In a further embodiment of in vitro assays, the target nucleicacid may be immobilised onto filter or polymer membranes or other typesof solid phases well-known in the art. The hybridisation conditions andmeans of detection may be selected according to the specific set-up asdescribed above.

[0304] In a further embodiment of the in vitro assay, an array of up to100 or even more different probes directed against different targetsequences may be immobilised onto a solid surface and hybridisation ofthe target sequences to all the probes is carried out simultaneously.The solid phase, the hybridisation conditions and means of detection maybe as described above. This allow for simultaneous detection oridentification of a range of parameters, i.e. species identification andresistance patterns.

[0305] The present probes further provide a method of diagnosinginfection by mycobacteria and a method for determining the stage of theinfection and the appropriate treatment by which methods one or moreoptionally labelled probes according to the invention are brought intocontact with a patient sample and the type of treatment and/or theeffect of a treatment is (are) evaluated.

[0306] Kits comprising at least one peptide nucleic acid probe asdefined herein are also part of the present invention. Such kit mayfurther comprise a detection system with at least one detecting reagentand/or a solid phase capture system.

DESCRIPTION OF SPECIFIC EMBODIMENTS

[0307] Examples of suitable Qs of adjacent moieties are given below.Peptide nucleic acid probes comprising such Qs will be suitable fordetecting mycobacteria, in particular mycobacteria of the MTC group ormycobacteria other than mycobacteria of the MTC group. The probes arewritten from left to right corresponding to from the N-terminal endtowards the C-terminal end. Suitable 0 subsequences for detecting 23Sand 16S rRNA as well as 5S rRNA of the MTC group are given below.Suitable Q subsequences for detecting 23S and 16S rRNA of mycobacteriaother than mycobacteria of the MTC group are further given below. The Qsubsequences include at least one nucleobase complementary to anucleobase selected from the positions given in parenthesis. The Qsubsequences are given as non-limiting examples of construction ofsuitable probe nucleobase sequences. It is to be understood that theprobes may comprise fewer or more peptide nucleic acid moieties thanindicated. MTC group (23S) AGA TGC GGG TAG GAG (selected from positions149-158 in FIG. 1A), (Seq ID no 1) TGT TTT CTC CTC CTA (selected frompositions 220-221 in FIG. 1A), (Seq ID no 2) ACT GCC TCT CAG CCG(selected from positions 328-361 in FIG. 1A and (Seq ID no 3) FIG. 1B),TGA TAC TAG GCA GGT (selected from positions 453-455 in FIG. 1B), (SeqID no 4) CGG ATT CAC AGC GGA (selected from positions 490-501 in FIG.1B), (Seq ID no 5) TCA CCA CCC TCC TCC (selected from positions 637-660in FIG. 1C), (Seq ID no 6) CCA CCC TCC TCC (selected from positions637-660 in FIG. 1C), (modified Seq ID no 6) TTA ACC TTG CGA CAT(selected from positions 706-712 in FIG. 1D), (Seq ID no 7) ACT ATT CACACG CGC (selected from positions 762-789 in FIG. 1D), (Seq ID no 8) CTCCGC GGT GAA CCA (selected from position 989 in FIG. 1D), (Seq ID no 9)GCT TTA CAC CAC GGC (selected from positions 1068-1072 in FIG. 1E), (SeqID no 10) ACG CTT GGG GGC CTT (selected from position 1148 in FIG. 1E),(Seq ID no 11) CCA CAC CCA CCA CAA (selected from positions 1311-1329 inFIG. 1E), (Seq ID no 12) CCG GTG GCT TCG CTG (selected from positions1361-1364 in FIG. 1F), (Seq ID no 13) ACT TGC CTT GTC GCT (selected fromposition 1418 in FIG. 1F), (Seq ID no 14) GAT TCG TCA CGG GCG (selectedfrom positions 1563-1570 in FIG. 1F), (Seq ID no 15) AAC TCC ACA CCC CCG(selected from positions 1627-1638 in FIG. 1G), (Seq ID no 16) ACT CCACAC CCC CGA (selected from positions 1627-1638 in FIG. 1G), (Seq ID no17) ACC CCT TCG CTT GAC (selected from positions 1675-1677 in FIG. 1G),(Seq ID no 18) CTT GCC CCA GTG TTA (selected from position 1718 in FIG.1G), (Seq ID no 19) CTC TCC CTA CCG GCT (selected from positions1734-1740 in FIG. 1H), (Seq ID no 20) GAT ATT CCG GTC CCC (selected frompositions 1967-1976 in FIG. 1H), (Seq ID no 21) ACT CCG CCC CAA CTG(selected from positions 2403-2420 in FIG. 1H), (Seq ID no 22) CTG TCCCTA AAC CCG (selected from positions 2457-2488 in FIG. 1I), (Seq ID no23) TTC GAG GTT AGA TGC (selected from positions 2457-2488 in FIG. 1I),(Seq ID no 24) GTC CCT AAA CCC GAT (selected from positions 2457-2488 inFIG. 1I), (Seq ID no 25) GGT GCA CCA GAG GTT (selected from positions2952-2956 in FIG. 1I), (Seq ID no 26) CTG GCG GGA CAA CTG (selected frompositions 2966-2969 in FIG. 1J), (Seq ID no 27) TTA TCC TGA CCG AAC(selected from positions 3000-3003 in FIG. 1J), (Seq ID no 28) GAC CTATTG AAC CCG (selected from positions 3097-3106 in FIG. 1J), (Seq ID no29) MTC group (16S) GAA GAG ACC TTT CCG (selected from positions 76-79in FIG. 2A), (Seq ID no 30) CAC TCG AGT ATC TCC (selected from positions98-101 in FIG. 2A), (Seq ID no 31) ATC ACC CAC GTG TTA (selected frompositions 136-136 in FIG. 2A), (Seq ID no 32) GCA TCC CGT GGT CCT(selected from positions 194-201 in FIG. 2B), (Seq ID no 33) CAC AAG ACATGC ATC (selected from positions 194-201 in FIG. 2B), (Seq ID no 34) TAAAGC GCT TTC CAC (selected from positions 222-229 in FIG. 2B), (Seq ID no35) GCT CAT CCC ACA CCG (selected from position 242 in FIG. 2B), (Seq IDno 36) CCG AGA GAA CCC GGA (selected from position 474 in FIG. 2C), (SeqID no 37) AGT CCC CAC CAT TAC (selected from positions 1136-1145 in FIG.2C), (Seq ID no 38) AAC CTC GCG GCA TCG (selected from positions1271-1272 in FIG. 2C), (Seq ID no 39) GGC TTT TAA GGA TTC (selected frompositions 1287-1292 in FIG. 2D), (Seq ID no 40) GAC CCC GAT CCG AAC(selected from position 1313 in FIG. 2D), (Seq ID no 41) CCG ACT TCA CGGGGT (selected from position 1334 in FIG. 2D), (Seq ID no 42) MTC group(5S) CCC AGG CCC ACT ATC (selected from positions 86-90 in FIG. 3), (SeqID no 43) Mycobacteria other than those of the MTO group (23S) GAT CAATGC TCG GTT (selected from positions 99-101 in FIG. 4A), (Seq ID no 44)TTC CCC GCG TTA CCT (selected from position 183 in FIG. 4A), (Seq ID no45) TTA CCC TGT TTA GGT (selected from positions 261-271 in FIG. 4A),(Seq ID no 46) GCA TGC GGT TTA GCC (selected from positions 281-284 inFIG. 40), (Seq ID no 47) TAC CCG GTT GTC CAT (selected from positions290-293 in FIG. 4B), (Seq ID no 48) GTA GAG CTG AGA CAT (selected frompositions 327-335 and 343-357 in (Seq ID no 49) FIG. 4B), GCC GTC CCAGGC CAC (selected from positions 400-405 in FIG. 40 and (Seq ID no 50)FIG. 40), CTC GGG TGT TGA TAT (selected from positions 453-462 in FIG.4C), (Seq ID no 51) ACT ATT TCA CTC CCT (selected from positions 587-599in FIG. 4C), (Seq ID no 52) ACG CCA TCA CCC CAC (selected from positions637-660 in FIG. 4D), (Seq ID no 53) CGA CGT GTG CGT GAG (selected frompositions 704-712 in FIG. 4D), (Seq ID no 54) ACT ACA CCC CAA AGG(selected from positions 763-789 in FIG. 4E), (Seq ID no 55) CAC GCT TTTACA CCA (selected from positions 1060-1074 in FIG. 4E), (Seq ID no 56)GCG ACT ACA CAT CCT (selected from positions 1177-1185 in FIG. 4E), (SeqID no 57) CGG CGC ATA ATC ACT (selected from positions 1259-1265 in FIG.4E), (Seq ID no 58) CCA CAT CCA CCG TAA (selected from positions1311-1327 in FIG. 4F), (Seq ID no 59) CGC TGA ATG GGG GAC (selected frompositions 1345-1348 in FIG. 4E), (Seq ID no 60) GGA GCT TCG CTG AAT(selected from positions 1361-1364 in FIG. 4G), (Seq ID no 61) CGG TCACCC GGA GCT (selected from positions 1361-1364 in FIG. 4G), (Seq ID no62) GGA CGC CCA TAC ACG (selected from positions 1556-1570 in FIG. 4G),(Seq ID no 63) GAA GGG GAA TGG TCG (selected from positions 1608-1613 inFIG. 4H), (Seq ID no 64) AAT CGC CAC GCC CCC (selected from positions1626-1638 in FIG. 4H), (Seq ID no 65) CAG CGA AGG TCC CAC (selected frompositions 1651-1659 in FIG. 4H), (Seq ID no 66) GTC ACC CCA TTG CTT(selected from positions 1675-1677 in FIG. 4H), (Seq ID no 67) ATC GCTCTC TAC GGG (selected from positions 1734-1741 in FIG. 4H), (Seq ID no68) GTG TAT GTG CTC GCT (selected from positions 1847-1853 in FIG. 4I),(Seq ID no 69) ACG GTA TTC CGG GCC (selected from positions 1967-1976 inFIG. 4I), (Seq ID no 70) GGC CGA ATC CCG CTC (selected from positions2006-2010 in FIG. 4I), (Seq ID no 71) AAA CAG TCG CTA CCC (selected frompositions 2025-2027 in FIG. 4I), (Seq ID no 72) CCT TAC GGG TTA ACG(selected from positions 2131-2132 in FIG. 4J), (Seq ID no 73) GAG ACAGTT GGG AAG (selected from positions 2252-2255 in FIG. 4J), (Seq ID no74) TGG CGT CTG TGC TTC (selected from positions 2396-2405 in FIG. 4Jand (Seq ID no 75) FIG. 4K), CGA CTC CAC ACA AAC (selected frompositions 2416-2420 in FIG. 4K), (Seq ID no 76) GAT AAG GGT TCG ACG(selected from positions 2474-2478 in FIG. 4K), (Seq ID no 77) ATC CGTTGA GTG ACA (selected from position 2687 in FIG. 4K), (Seq ID no 78) CAGCCC GTT ATC CCC (selected from position 2719 in FIG. 4K), (Seq ID no 79)AAC CTT TGG GAC CTG (selected from position 2809 in FIG. 4L), (Seq ID no80) TAA AAG GGT GAG AAA (selected from positions 3062-3068 in FIG. 4L),(Seq ID no 81) GTC TGG CCT ATC AAT (selected from positions 3097-3106 inFIG. 4L), (Seq ID no 82) Mycobacteria other than those of the MTC group(16S) AGA TTG CCC ACG TGT (selected from positions 135-136 in FIG. 5A),(Seq ID no 83) AAT CCG AGA AAA CCC (selected from positions 472-475 inFIG. 5A), (Seq ID no 84) GCA TTA CCC GCT GGC (selected from positions1136-1144 in FIG. 5A), (Seq ID no 85) TTA AAA GGA TTC GCT (selected frompositions 1287-1292 in FIG. 5B), (Seq ID no 86) AGA CCC CAA TCC GAA(selected from position 1313 in FIG. 5B), (Seq ID no 87) GAC TCC GAC TTCATG (selected from position 1334 in FIG. 5B), (Seq ID no 88) Drugresistance 23S-mediated macrolide resistance (M. avium) GTC TTT TCG TCCTGC (wild-type) (selected from positions 2568-2569 in FIG. 6), (Seq IDno 89) GTC TTA TCG TCC TGC (selected from positions 2568 in FIG. 6),(Seq ID no 90) GTC TTC TCG TCC TGC (selected from positions 2568 in FIG.6), (Seq ID no 91) GTC TTG TCG TCC TGC (selected from positions 2568 inFIG. 6), (Seq ID no 92) GTC TAT TCG TCC TGC (selected from positions2568 in FIG. 6), (Seq ID no 93) GTC TCT TCG TCC TGC (selected frompositions 2568 in FIG. 6), (Seq ID no 94) GTC TGT TCG TCC TGC (selectedfrom positions 2568 in FIG. 6), (Seq ID no 95) 16S-mediated streptomycinresistance (M. tuberculosis) TTG GCC GGT GCT TCT (wild-type) (selectedfrom positions 452 in FIG. 7), (Seq ID no 96) TTG GCC GGT ACT TCT(selected from positions 452 in FIG. 7), (Seq ID no 97) TTG GCC GGT CCTTCT (selected from positions 452 in FIG. 7), (Seq ID no 98) TTG GCC GGTTCT TCT (selected from positions 452 in FIG. 7), (Seq ID no 99) ACC GCGGCT GCT GGC (wild-type) (selected from positions 473-477 in FIG. 7) (SeqID no 100) ACC GCG GCT ACT GGC (selected from positions 473 in FIG. 7),(Seq ID no 101) ACC GCG GCT CCT GGC (selected from positions 473 in FIG.7), or (Seq ID no 102) ACC GCG GCT TCT GGC (selected from positions 473in FIG. 7), (Seq ID no 103) CGG CAG CTG GCA CGT (selected from positions474 in FIG. 7), (Seq ID no 104) CGG CCG CTG GCA CGT (selected frompositions 474 in FIG. 7), (Seq ID no 105) CGG CTG CTG GCA CGT (selectedfrom positions 474 in FIG. 7), (Seq ID no 106) CGT ATT ACC GCA GCT(selected from positions 477 in FIG. 7), (Seq ID no 107) CGT ATT ACC GCCGCT (selected from positions 477 in FIG. 7), (Seq ID no 108) CGT ATT ACCGCT GCT (selected from positions 477 in FIG. 7), (Seq ID no 109) TTC CTTTGA GTT TTA (wild-type) (selected from positions 865-866 in FIG. 7),(Seq ID no 110) TTC CTT TAA GTT TTA (selected from positions 665 in FIG.7), (Seq ID no 111) TTC CTT TCA GTT TTA (selected trom positions 865 inFIG. 7), (Seq ID no 112) TTC CTT TTA GTT TTA (selected from positions865 in FIG. 7), (Seq ID no 113) TTC CTT ACA GTT TTA (selected trompositions 866 in FIG. 7), (Seq ID no 114) TTC CTT CGA GTT TTA (selectedfrom positions 866 in FIG. 7), (Seq ID no 115) TTC CTT GGA GTT TTA(selected from positions 866 in FIG. 7), (Seq ID no 116) Other examplesof suitable Q subsequences are given below CAT GTG TCC TGT GGT and (SeqID no 117) CGT CAG CCC GAG AAA (Seq ID no 118)

[0308] selected so as to be complementary to M. gordonae 16S rRNA(positions 174-188 and 452-466, respectively, of GenBank entryGB:MSGRR16SI, accession no. M29563). These positions correspond topositions 192-206 and 473-487, respectively, of the alignments shown inFIG. 2 and 5. Probes having this or a similar nucleobase sequence aresuitable for detecting M. gordonae. CAC TAC ACA CGC TCG, and (Seq ID no119) TGG CGT TGA GGT TTC (Seq ID no 120)

[0309] selected so as to be complementary to positions 781-795 and2369-2383, respectively, of M. kansasii 23S rRNA (GenBank entryMK23SRRNA accession number Z17212). These positions correspond topositions 774-794 and 2398-2412, respectively, of the alignments shownin FIGS. 1 and 4. Probes having this or a similar nucleobase sequenceare suitable for detecting M. kansasii.

[0310] Precursor rRNA

[0311] AAC ACT CCC TTT GGA (Seq ID no 123)

[0312] A peptide nucleic acid probe having the above-indicatednucleobase sequence is directed to M. tuberculosis precursor rRNA. Theprobe is complementary to positions 602 to 616 of GenBank accessionnumber X58890.

[0313] Especially, probes based on those nucleobase sequences withsequence identification numbers Seq ID no 62, 79 and 80 (and otherprobes selected from positions 1361-1364 in FIG. 1F, 2719 in FIG. 4K and2809 in FIG. 4L) are suitable for detecting M. avium. Probes based onthe nucleobase sequence with sequence identification number Seq ID no 55(and other probes selected from positions 763-789 in FIG. 4E) aresuitable for detecting M. avium, M. intracellulare and M. scrofulaceumas a group (the organisms termed the MAIS group of mycobacteria). Inaddition, probes based on the nucleobase sequences with sequenceidentification numbers Seq ID no 77 and 81 are suitable for detecting M.avium, M. intracellulare and M. paratuberculosis as a group.

[0314] The invention is further illustrated by the non-limiting examplesgiven below.

EXAMPLES Example 1

[0315] Mycobacterium species (M. bovis and M. intracellulare) 23S rDNAwere partly amplified by PCR, and the PCR products were sequenced (bothstrands) using Cy5-labelled oligonucleotide primers (DNA Technology,Aarhus, Denmark) and the 7-deaza-dGTP Thermo Sequenase cycle sequencingkit from Amersham, Little Chalfont, England. Sequences were read usingan ALFexpress automated sequencer and ALFwin (version 1.10) softwarefrom Pharmacia Biotech, Uppsala, Sweden. M. bovis and M. intracellulare23S rRNA sequences are included at the following positions of the 23SrDNA sequence alignments: positions 681-729 (FIGS. 1C and 4D), positions761-800 (FIGS. 1D and 4E), positions 2401-2440 (FIGS. 1H and 4K),positions 2441-2480 (FIGS. 1I and 4K), positions 2481-2520 (FIG. 1I),positions 3041-3080 (FIG. 4L), and positions 3081-3120 (FIGS. 1J and4L).

Example 2

[0316] Sequence alignments (see FIGS. 1 to 5) of 23S, 16S and 5S rDNA ofmycobacteria of the MTC group, and 23S and 16S rDNA of mycobacteriaother than those of the MTC group (MOTT) were done using the Megalign(version 3.12) alignment tool from DNASTAR (Madison, Wis., USA). Up toone hundred sequences were aligned at a time.

[0317] Peptide nucleic acid probes in which the nucleobase sequence wascomplementary to distinctive mycobacterial rRNA were designed with dueregard to secondary structures using the PrimerSelect program (version3.04) from DNASTAR. As a control of sequence specificity, all probesequences were subsequently matched with the GenBank and EMBL databasesusing BLAST sequence similarity searching at the National Center forBiotechnology Information (http://www.ncbi.nIm.nih.gov).

[0318] As examples, the following sequences were selected: MTC 235 TCACCA CCC TCC TCC (Seq ID no 6) CCA CCC TCC TCC (modified Seq ID no 6) ACTATT CAC ACG CGC (Seq ID no 8) CCA CAC CCA CCA CAA (Seq ID no 12) AAC TCCACA CCC CCG (Seq ID no 16) ACT CCA CAC CCC CGA (Seq ID no 17) ACT CCGCCC CAA CTG (Seq ID no 22) CTG TCC CTA AAC CCG (Seq ID no 23) TTC GAGGTT AGA TGC (Seq ID no 24) GTC CCT AAA CCC GAT (Seq ID no 25) GAC CTATTG AAC CCG (Seq ID no 29) MTC 16S GCA TCC CGT GGT CCT (Seq ID no 33)CAC AAG ACA TGC ATC (Seq ID no 34) GGC TTT TAA GGA TTC (Seq ID no 40)MOTT 23S GAT CAA TGC TCG GTT (Seq ID no 44) CGA CTC CAC ACA AAC (Seq IDno 76) MOTT 16S GCA TTA CCC GCT GGC (Seq ID no 85) Drug resistance GTCTTA TCG TCC TGC (Seq ID no 90) GTC TTC TCG TCC TGC (Seq ID no 91) GTCTTG TCG TCC TGC (Seq ID no 92) GTC TAT TCG TCC TGC (Seq ID no 93) GTCTCT TCG TCC TGC (Seq ID no 94) GTC TGT TCG TCC TGC (Seq ID no 95)Precursor rRNA AAC ACT CCC TTT GGA (Seq ID no 123) Non-sense probes GTCCGT GAA CCC GAT (Seq ID no 121) TAC GCT CTT TGA GCT (Seq ID no 122)

Example 3

[0319] Peptide nucleic acid probes were synthesised using an Expedite8909 Nucleic Acid Synthesis System purchased from PerSeptive Biosystems(Framingham, USA). The peptide nucleic acid probes were terminated withtwo p-alanine molecules or with one or two lysine molecule(s) and,before cleavage from the resin, labelled with 5-(or6)-carboxyfluorescein (Flu) or rhodamine (Rho) at the β-amino group ofalanine (peptide label) or ε-amino group of lysine (peptide label),respectively. Probes were purified using reverse phase HPLC at 50° C.and characterised using a G2025 A MALDI-TOF MS instrument (HewlettPackard, San Fernando, Calif., USA). Molecular weights determined werewithin 0.1% of the calculated molecular weights.

[0320] The following labelled peptide nucleic acid probes weresynthesised: MTC 23S Lys(Flu)-Lys(Flu)-TCA CCA CCC TCC TCC-NH₂ (OK446/modified Seq ID no 6) Lys(Flu)-Lys(Flu)-CCA CCC TCC TCC-NH₂ (OK575/modified Seq ID no 6) Lys(Flu)-Lys(Flu)-ACT ATT CAC ACG CGC-NH₂ (OK447/modified Seq ID no 8) Lys(Flu)-ACT ATT CAC ACG CGC-NH₂ (OK688/modified Seq ID no 8) Lys(Flu)-Lys(Flu)-CCA CAC CCA CCA CAA-NH₂ (OK448/modified Seq ID no 12) Lys(Flu)-Lys(Flu)-AAC TCC ACA CCC CCG-NH₂ (OK449/modified Seq ID no 16) Lys(Flu)-Lys(Flu)-ACT CCA CAC CCC CGA-NH₂ (OK309/modified Seq ID no 17) Lys(Flu)-Lys(Flu)-ACT CCG CCC CAA CTG-NH₂ (OK450/modified Seq ID no 22) Lys(Flu)-Lys(Flu)-CTG TCC CTA AAC CCG-NH₂ (OK305/modified Seq ID no 23) Lys(Flu)-Lys(Flu)-TTC GAG GTT AGA TGC-NH₂ (OK306/modified Seq ID no 24) Lys(Flu)-TTC GAG GTT AGA TGC-NH₂ (OK682/modified Seq ID no 24) Lys(Flu)-Lys(Flu)-GTC CCT AAA CCC GAT-NH₂ (OK307/modified Seq ID no 25) Lys(Flu)-GTC CCT AAA CCC GAT-NH₂ (OK654/modified Seq ID no 25) Lys(Flu)-GAC CTA TTG AAC CCG-NH₂ (OK660/modified Seq ID no 29) MTC 16S Lys(Flu)-Lys(Flu)-Gly-GCA TCC CGT GGTCCT-NH₂ (OK 223/modified Seq ID no 33) Lys(Flu)-Lys(Flu)-CAC AAG ACA TGCATC-NH₂ (OK 310/modified Seq ID no 34) Lys(Flu)-CAC AAG ACA TGC ATC-NH₂(OK 655/modified Seq ID no 34) Lys(Flu)-GGC TTT TAA GGA TTC-NH₂ (OK689/modified Seq ID no 40) Lys(Rho)-GGC TTT TAA GGA TTC-NH₂ (OK702/modified Seq ID no 40) MOTT 23S Flu-β-Ala-β-Ala-GAT CAA TCG TCGGTT-NH₂ (OK 624/modified Seq ID no 44) Flu-β-Ala-β-Ala-CGA CTC GAG ACAAAC-NH₂ (OK 612/modified Seq ID no 76) MOTT 16S Flu-β-Ala-β-Ala-GCA TTACCC GCT GGC-NH₂ (OK 623/modified Seq ID no 85) Drug resistanceLys(Flu)-GTG TTT TCG TCC TGC-NH₂ (OK 745/modified Seq ID no 89)Lys(Rho)-GTC TTA TCG TCC TGC-NH₂ (OK 746/modified Seq ID no 90)Lys(Rho)-GTC TTG TCG TCC TGC-NH₂ (OK 746/modified Seq ID no 91)Lys(Rho)-GTC TTG TCG TCC TGC-NH₂ (OK 746/modified Seq ID no 92)Lys(Rho)-GTC TAT TCG TCC TGC-NH₂ (OK 747/modified Seq ID no 93)Lys(Rho)-GTC TCT TCG TCC TGC-NH₂ (OK 747/modified Seq ID no 94)Lys(Rho)-GTC TGT TCG TCC TOG-NH₂ (OK 747/modified Seq ID no 95)Precursor rNA Lys(Flu)-AAC ACT CCC TTT GGA-NH₂ (OK 749/modified Seq IDno 123) Reduction of non-specific binding GTC CGT GAA CCC GAT-NH₂ (OK507/modified Seq ID no 121) Gly-TAG GCT CTT TGA GCT-NH₂ (OK 714/modifiedSeq ID no 122)

Example 4

[0321] Initially the ability of the peptide nucleic acid probes to reactwith target sequences of mycobacterial rRNA was tested by dot blotcarried out with rRNA from M. bovis BCG, M. avium and E. coli.

[0322]M. bovis BCG (Statens Serum Institut, Denmark) and M.intracellulare (kindly provided by Statens Serum Institut) were grown inDubos broth (Statens Serum Institut) or on Löwenstein-Jensen slants(Statens Serum Institut) at 37° C. RNA was isolated from the bacterialcells using TRI-reagent (Sigma) following manufacture's directions. E.coli rRNA was purchased from Boehringer Mannheim, Germany.

[0323] 200 ng M. bovis RNA, M. intracellulare RNA and E. coli rRNA weredotted onto membranes (Schleicher & Schüel, NY 13 N), and the membraneswere dried and fixed under UV light for 2 minutes.

[0324] Protocol for Dot Blot Assay

[0325] Each of the probes (70 nM probe in hybridisation solution (50 mMTris, 10 mM NaCl, 10% (w/v) Dextran sulphate, 50% (v/v) glycerol, 5 mMEDTA, 0.1% (w/v) sodium pyrophosphate, 0.2% (w/v) polyvinylpyrrolidone,0.2% (w/v) Ficoll, pH 7.6.)) were spotted onto a membrane. Hybridisationwas continued for 1.5 hours at 55 or 65° C., respectively. The membraneswere rinsed 2 times for 15 minutes in 2×SSPE buffer (1 x SSPE: 0.15 MNaCl, 10 mM sodium phosphate, 1 mM EDTA, pH 7.4) containing 0.1% SDS atambient temperature, and subsequently 2 times for 15 minutes in 0.1×SSPEbuffer containing 0.1% SDS at 55 or 65° C. (see Table 1). The membranewas blocked with 0.5% (w/v) casein dissolved in 0.5M NaCl, 0.05MTris/HCl pH 9.0. Thereafter, the membranes were incubated for 1 hourwith rabbit-anti FITC antibody labelled with alkaline phosphatase (AP)(DAKO K0046 vial A) diluted 1:2000 in 0.5% casein dissolved in 0.5MNaCl, 0.05M Tris/HCl pH 9.0. After incubation, the membranes were washed3 times 5 minutes with TST buffer (0.05M Tris, 0.5M NaCl, 0.5% (w/v)Tween 20®, pH 9) at ambient temperature. Bound probes were visualisedfollowing standard procedures using BCIP/NBT, and the visualisation wasstopped by incubation for 10 minutes with 10 mM EDTA. The blot was driedat 50° C.

[0326] The results are given in Table 1 below. TABLE 1 E. coli M. bovisBCG M. intracellulare rRNA RNA RNA Probe 55° C. 65° C. 55° C. 65° C. 55°C. 65° C. OK 305 negative negative positive positive negative weak OK307 negative negative positive positive negative weak OK 309 negativenegative positive positive negative weak OK 223 negative negativepositive positive nd nd OK 310 negative negative negative positivenegative negative

[0327] The results indicate that all five peptide nucleic acid probesare capable of hybridising to target sequence of M. bovis BCG rRNA (as arepresentative of the MTC group), whereas no hybridisation to E. colirRNA (as a representative of organisms other than mycobacteria) and nodetectable hybridisation to M. intracellulare rRNA were observed (as arepresentative of the MOTT group).

Example 5

[0328] This example illustrates the ability of the peptide nucleic acidprobes to penetrate the mycobacterial cell wall and subsequentlyhybridise to target sequence of mycobacteria of the MTC group and notmycobacteria of the MOTT group, in particular not mycobacteria of theMAC group, or Neisseria gonorrhoeae, by fluorescence in situhybridisation (FISH).

[0329] Preparation of Bacterial Slides

[0330]M. bovis BCG (Statens Seruminstitut, Denmark), M. avium (kindlyprovided by Statens Seruminstitut, Denmark), and M. intracellulare(kindly provided by Statens Seruminstitut, Denmark) were grown in Dubosbroth (Statens Seruminstitut, Denmark) or on Löwenstein-Jensen slants(Statens Seruminstitut, Denmark) at 37° C. N. gonorrhoeae (StatensSeruminstitut, Denmark) was grown on chocolate agar (StatensSeruminstitut, Denmark) at 37° C. with additional 5% CO₂.

[0331] Cultures were smeared onto microscope slides and fixed accordingto standard procedures. Prior to the hybridisation, the smears wereimmersed into 80% ethanol for 15 minutes, and subsequently rinsed withwater and air dried. This step is not essential for the followinghybridisation step, but it is anticipated that it will kill any viablemycobacteria on the slides, and may further serve as an additionalfixation step.

[0332] Protocol for Fluorescence in situ Hybridisation (FISH)

[0333] 1. The bacterial slide was covered with a hybridisation solutioncontaining the probe in question.

[0334] 2. The slide was incubated in a humid incubation chamber at 45°C. or 55° C. for 90 minutes.

[0335] 3. The slide was washed 25 minutes at 45° C. or 55° C. inprewarmed wash solution (5 mM Tris, 145 mM NaCl, pH 10) followed by 30seconds in water.

[0336] 4. The slide was dried and mounted with IMAGEN Mounting Fluid(DAKO, Copenhagen, Denmark)

[0337] The hybridisation solution contains 50 mM Tris, 10 mM NaCl, 10%(w/v) Dextran sulphate, 30% (v/v) formamide, 0.1% (v/v) Triton X-100®, 5mM EDTA, 0.1% (w/v) sodium pyrophosphate, 0.2% (w/v)polyvinylpyrrolidone, 0.2% (w/v) Ficoll, pH 7.6.

[0338] Whenever possible, the applied equipment was heat-treated, andsolutions were exposed to 1 μl/ml diethylpyrocarbonate (Sigma ChemicalCo.) in order to inactivate nucleases.

[0339] Microscopically examinations were conducted using a fluorescencemicroscope (Leica, Wetzlar, Germany) equipped with a 100×/1.20 waterobjective, a HBO 100 W lamp and a FITC filter set. Mycobacteria wereidentified as fluorescent, 1-10 μm slender, rod-shaped bacilli.

[0340] Fluorescein-labelled peptide nucleic acid probes targeting 23SrRNA of the mycobacteria of the MTC group (OK 306, OK 309, OK 446, OK449) and 16S rRNA of the mycobacteria of the MTC group (OK 310) weretested. Individual probe concentrations and incubation temperatures arelisted together with the results in Table 2 and 3. TABLE 2 OK 306 OK 309OK 446 OK 449 250 nM 250 nM 500 nM 500 nM 45° C. 45° C. 55° C. 55° C. M.bovis BCG positive positive positive positive M. avium negative negativenegative negative M. intracellulare negative negative not determined notdetermined N. gonorrhoeae negative negative not determined notdetermined

[0341] TABLE 3 OK 447 OK 310 OK 306/OK 310 1 μM 250 nM 500/500 nM 55° C.45° C. 55° C. M. bovis BCG positive positive positive M. avium negativenegative negative M. intracellulare not determined negative negative N.gonorrhoeae not determined negative not determined

[0342] It can be concluded that the probes are able to penetrate themycobacterial cell wall of mycobacterium cultures and subsequentlyhybridise to target rRNA sequence. This makes possible the developmentof fluorescence in situ hybridisation (FISH) protocols for specificdetection of mycobacteria.

Example 6

[0343] Test of Probes on Clinical Smears of Sputum

[0344] The ability of the peptide nucleic acid to penetrate the cellwall of mycobacteria of the MTC group in clinical samples was tested onsmears of sputum from suspected cases of tuberculosis (kindly providedby Division of Microbiology, Ramathibodi Hospital, Bangkok, Thailand) byfluorescence in situ hybridisation (FISH). Smears from the same patientwere initially evaluated positive by Ziehl-Neelsen staining, which showsonly the presence of acid fast bacilli, not whether these aremycobacteria of the MTC group.

[0345] Fluorescein-labelled peptide nucleic acid probes targeting 23SrRNA of the mycobacteria of the MTC group (OK 306, OK 446, OK 449) and16S rRNA of the mycobacteria of the MTC group (OK 310) were used.Furthermore, a random peptide nucleic acid probe (a 15-mer wherein eachposition may be A, T. C or G (obtained from Millipore Corporation,Bedford, Mass., USA) was added to the hybridisation solution in order toincrease the signal-to-noise ratio. FISH was carried out at 55° C. asdescribed in Example 5. Applied probe concentrations are listed togetherwith the results in Table 4 and 5. TABLE 4 Sample OK 446/Random OK449/Random Ziehl-Neelsen number 1 μM/50 μM 1 μM/50 μM staining 285Positive Positive 4+ 335 Positive Eq. 2+ 345 Positive Positive 3+ 224Positive Positive 3+ 297 Negative Eq. 2+ 179 Negative Negative 4+ 247Negative Negative 2+ 255 Positive Positive 2+ 202 Eq. Positive 2+

[0346] TABLE 5 Sample OK 306/OK 310 Ziehl-Neelsen number 500/500 nMstaining 213 Positive 4+ 292 Positive 4+ 159 Positive 3+ 287 Positive 3+

[0347] Smears stained by Ziehl-Neelsen staining were examined with a100× objective and scored according to the following method: −: 0bacilli, +/−: 1-200 per 300 fields, 2+: 1-9 per 10 fields, 3+:1-9 perfield, 4+: >9 per field.

[0348] Positive:Several mycobacteria were identified in the smear.Negative: No fluorescent mycobacteria were identified in the smear. Eq:Few (1-3) fluorescent mycobacteria were identified in the smear.

[0349] It appears from the table that the peptide nucleic acid probesare able to penetrate and subsequently hybridise to target sequence ofmycobacteria of the MTC-group in AFB-positive sputum smears. The factthat not all AFB-positive sputum smears are found positive with appliedprobes indicate that not all AFB-positive sputum smears containsmycobacteria of the MTC-group.

Example 7

[0350] The reactivity and specificity of selected peptide nucleic acidprobes for detecting mycobacteria of the MTC group as well as probes fordetecting mycobacteria of the MOTT group were evaluated by fluorescencein situ hybridisation (FISH) on control smears prepared from cultures ofdifferent mycobacterium species. The mycobacterium species were selectedso as to be representative for the mycobacterium genus as well as toinclude clinically relevant species.

[0351] M. tuberculosis (ATCC 25177), M. bovis BCG (ATCC 35734), M.intracellulare (ATCC 13950), M. avium (ATCC 25292), M. kansasi(ATCC12479), M. gordonae (ATCC 14470), M. scrofulaceum (ATCC 19981), M.abscessus (ATCC19977), M. marinum (ATCC 927), M. simiae (ATCC 25575), M.szulgai (ATCC 35799), M. flavescens (ATCC 23033), M. fortuitum (ATCC43266) and M. xenopi (ATCC19250) were grown at Dubos broth (StatensSerum Institut) at 37° C. with the exception of M. marinum which wasgrown at 32° C.

[0352] Smears were prepared as described in Example 5. FISH was carriedout as described below.

[0353] Protocol for Fluorescence in situ Hybridisation (FISH)

[0354] 1. The bacterial slide was covered with a hybridisation solutioncontaining the probe in question.

[0355] 2. The slide was incubated in a humid incubation chamber at 55°C. for 90 minutes.

[0356] 3. The slide was washed 30 minutes at 55° C. in prewarmed washsolution (5 mM Tris, 15 mM NaCl, 0.1% (v/v), Triton X-100®, pH 10)followed by 30 seconds in water.

[0357] 4. The slide was dried and mounted with IMAGEN Mounting Fluid(DAKO, Copenhagen, Denmark)

[0358] The hybridisation solution contained 50 mM Tris, 10 mM NaCl, 10%(w/v) Dextran sulphate, 30% (v/v) formamide, 0.1% (v/v) Triton X-100®, 5mM EDTA, 0.1% (w/v) sodium pyrophosphate, 0.2% (w/v)polyvinylpyrrolidone, and 0.2% (w/v) Ficoll, pH 7.6. To avoidnon-specific binding of the labelled peptide nucleic acid probe, 1-5 μMof non-labelled, non-sense peptide nucleic acid probe (OK 507/modifiedSeq ID no 121 and/or OK 714/modified Seq ID no 122) was added to thehybridisation solution.

[0359] Whenever possible, the applied equipment was heat-treated, andsolutions were exposed to 1 μl/ml diethylpyrocarbonate (Sigma ChemicalCo.) in order to inactivate nucleases.

[0360] Microscopic examinations were conducted using a fluorescencemicroscope (Leica, Wetzlar, Germany) equipped with a 100×/1.30 oilobjective, a HBO 100 W lamp and a FITC/TRITC dual band filter set.Mycobacteria were identified on basis of both fluorescence (strong,medium, weak, no) and morphology (1-10 μm slender, rod-shaped bacilli.Mycobacteria of the MOTT group may appear pleomorphic, ranging inappearance from long rods to coccoid forms)

[0361] Probe concentrations are listed together with the results inTable 6 and 7 (probes targeting mycobacteria of the MTC group) and Table8 (probes targeting to mycobacteria of the MOTT group). TABLE 6 OK 450OK 682 OK 689 OK 688 OK 660 25 nM 100 nM 100 nM 250 nM 100 nM M.tuberculosis +++ +++ +++ +++ +++ M. bovis BCG +++ +++ +++ +++ +++ M.intracellulare − − − − − M. avium − − − − − M. kansasii ++ − − − − M.gordonae − − − − − M. scrofulaceum +++ − − − − M. abscessus − − − − + M.marinum +++ − + + +++ M. simiae − − − − − M. szulgai +++ − − − − M.flavescens − ++ − − − M. fortuitum − + − − − M. xenopi − ++ − − −

[0362] TABLE 7 OK 655 OK 448 OK 654 OK 446 Mycobacteria 150 nM 50 nM 100nM 25 nM M. tuberculosis +++ +++ +++ +++ M. bovis BCG +++ +++ +++ +++ M.intracellulare − − − − M. avium − − − − M. kansasii − − − − M. gordonae− − − − M. scrofulaceum − − − − M. abscessus − − + − M. marinum − − ++++ M. simiae − − − − M. szulgai − − − − M. flavescens − − − − M.fortuitum − − − − M. xenopi − − − −

[0363] TABLE 8 OK 612 OK 624 OK 623 Mycobacteria 100 nM 100 nM 100 nM M.tuberculosis − − − M. bovis BCG − − − M. intracellulare − ++ ++ M. avium+++ +++ +++ M. kansasii − − +++ M. gordonae − ++ ++ M. scrofulaceum − ++++ M. abscessus − ++ +++ M. marinum − − − M. simiae − ++ +++ M. szulgai− − +++ M. flavescens − − − M. fortuitum − ++ − M. xenopi − − −

[0364] Each of probes indicated in Table 6, 7 and 8 was furtherinvestigated with regard to hybridisation to other common respiratorybacteria, namely Corynebacterium spp., Fusobacterium nucleatum,Haemophilus influenzae, Klebsiella pneumoniae, Pseudomonas aeruginosa,Propionibacterium acnes, Streptococcuc pneumoniae, Staphylococcusaureus, Brahamella catarrahalis, Escherichia coli, Neisseria spp.,Actinobacter calcoaceticus, Actinomyces spp., Enterobacter aerogenes,Proteus mirabilis, Pseudomonas maltophilia, Streptocussuc viridans, andNorcardia asteroides. No cross-hybridisation was observed byfluorescence in situ hybridisation to any of these bacteria in the caseof OK 682, OK 654, OK 655, OK 688, OK 660, OK 612, OK 624 and OK 623.Some cross-reactivity was observed in the case of OK 446 (to P. acnes),OK 448 (to P. acnes and B. catarrhalis), and OK 450 (to P. acnes and B.catarrhalis).

[0365] Table 6 and 7 shows that none of the MTC probes cross-react withM. intracellulare and/or M. avium, but indeed strongly with M.tuberculosis and M. bovis BCG. As shown in Table 8, both OK 624 and OK623 hybridise to M. intracellulare and M. avium which are both membersof the MAC group, whereas none of them hybridise to M. tuberculosis orM. bovis BCG. OK 612 hybridises to M. avium only. It should be notedthat the aligned sequence of M. intracellulare has just one nucleobasedifference to the target sequence of M. avium, see FIG. 4K.

[0366] The data support the use of the methodology described in claim 3and 4 and exemplified in Example 2 for design of peptide nucleic acidprobes that are capable of hybridising to target sequence of one or moremycobacterium species and not to other mycobacterium species having atleast one nucleobase difference to the target sequence.

Example 8

[0367] To study the usefulness of the peptide nucleic acid probes indistinguishing between mycobacteria of the MTC group and mycobacteria ofthe MOTT group, the probes were tested on smears ofmycobacterium-positive cultures prepared from 34+28 clinical samples(sputum samples, other respiratory samples and extrapulmonary samples)from individuals suspected of tuberculosis or other mycobacterialinfections (kindly provided by the Mycobacterium Department, StatensSerum Institut, Denmark). Complex/species identification data obtainedwith the AccuProbe tests from Gen-Probe Inc., USA were available foreach sample.

[0368] Table 9 shows the results obtained with four different peptidenucleic acid probes targeting mycobacteria of the MTC group (OK 682, OK660, OK 688 and OK 689) and one probe targeting mycobacteria of the MOTTgroup (OK 623), and Table 10 shows the results obtained with two peptidenucleic acid probes targeting mycobacteria of the MOTT group (OK 623 andOK 612) and a mixture of two probes targeting mycobacteria of the MTCgroup (OK 688 and OK 689). Data are arranged according to the resultsobtained by AccuProbe. Sample preparation, hybridisation andvisualisation were performed as described in Example 7. TABLE 9 OK 623OK 682 OK 660 OK 688 OK 689 Complex/ 25 nM 100 nM 100 nM 250 nM 100 nMspecies (n) n_(p) n_(p) n_(p) n_(p) n_(p) MTC (23) 0 23 23 23 23 M.avium (5) 5 0 0 0 0 M. gordonae (3) 3 0 0 0 0 Unknown (3) 3 0 0 0 0

[0369] TABLE 10 OK 623 OK 612 OK 688/OK 689 Complex/ 25 nM 100 nM 50nM/50 nM species (n) n_(p) n_(p) n_(p) MTC (17) 0 16 M. avium (2) 2 2 0M. gordonae (4) 3 0 0 Unknown (5) 5 0 0

[0370] The results shown in Table 9 are in conformity with thecomplex/species identification performed with the AccuProbe tests, andthus confirm that peptide nucleic acid probes can be used to determinewhether an infection is caused by mycobacteria of the MTC group or bymycobacteria of the MOTT group.

[0371] From the results in Table 10, it can be seen that it is possibleto differentiate between mycobacteria of the MTC group and mycobacteriaof the MOTT group with 100% specificity and 91-94% sensitivity relativeto results obtained by the AccuProbe tests. Furthermore, OK 612 is verysuitable for specific identification of M. avium among those beingpositive for mycobacteria of the MOTT group as the result is positive inthe case of M. avium and negative in the other cases of mycobacteria ofthe MOTT group.

Example 9

[0372] Direct Detection of Mycobacteria in Clinical Smears of Sputum

[0373] This example demonstrates the ability of the peptide nucleic acidto detect and identify mycobacteria directly in AFB-positive sputumsamples from suspected cases of tuberculosis (kindly provided byDivision of Microbiology, Ramathibodi Hospital, Bangkok, Thailand) andsuspected cases of other mycobacterial infections (kindly provided byClinical Microbiology Dept., Rigshospitalet, Copenhagen, Denmark) byFISH is shown.

[0374] The clinical smears were prepared according to the proceduredescribed in Example 5, and FISH was performed as described in Example7. The results are shown in Table 11. TABLE 11 OK 623 OK 654 OK 655 OK682 OK 688 OK 689 Sample no. 25 nM 100 nM 150 nM 100 nM 250 nM 100 nM 1− ++ ++ ++ ++ ++ 175 − ++ nd nd ++ ++ 459 − − nd nd − − 166 − − − nd − −268 − ++ ++ ++ ++ ++ 34267 ++ − − − − −

[0375] It appears from examples in Table 11 that AFB-positive sputumsmears were evaluated positive for mycobacteria of the MTC group (samplenumbers 1, 175, and 268), positive for mycobacteria of the MOTT group(sample number 37267), or negative for mycobacteria (sample numbers. 459and 166) by the applied probes. Thus, PNA-probes are useful reagents forspecific identification of mycobacteria directly in sputum smears byfluorescence in situ hybridisation. AFB-positive sputum samples that arenegative with all probes may be explained in three ways: a) the samplemay contain mycobacteria not detected by the probes, e.g. M. fortuitum,b) the sample may contain other acid-fast bacteria than mycobacteria, orc) the mycobacteria in the sample lack or have a strongly reducedcontent of rRNA due to for example antibiotic treatment.

[0376] In conclusion, direct identification of mycobacteria insmear-positive sputum samples by peptide nucleic acid-based fluorescencein situ hybridisation combines simplicity and morphological advantagesof current staining methods with concominant species identification, andwill thus allow clinical microbiology laboratories to benefit from theadvantages offered by molecular techniques to provide crucialinformation pertaining to therapy and patient management.

Example 10

[0377] This example demonstrates simultaneous detection andidentification of mycobacteria of the MTC group and mycobacteria of theMOTT group using differently labelled probes targeting mycobacteria ofthe MTC group and mycobacteria of the MOTT group, respectively, byfluorescence in situ hybridisation.

[0378] Control smears of different mycobacterium species were preparedas described in Example 5. In addition, smears containing a mixture ofM. tuberculosis and M. avium were prepared (Table 8, last row). FISH wasperformed as described in Example 7.

[0379] A rhodamine-labelled peptide nucleic acid probe targeting 16SrRNA of mycobacteria of the MTC group (OK 702) and afluorescein-labelled peptide nucleic acid probe targeting 16S rRNA ofmycobacteria of the MOTT group (OK 623) were applied simultaneously inthe concentrations listed in Table 12 together with the results. TABLE12 OK 623/OK 702 Mycobacterium species 25/250 nM M. tuberculosis−(G)/+++(R) M. bovis BCG −(G)/+++(R) M. avium +++(G)/−(R) M.intracellulare +++(G)/−(R) M. kansasii +++(G)/−(R) M. avium/M.tuberculosis +++(G)/+++(R)

[0380] Mycobacteria of the MTC group, i,e. M. tuberculosis and M. bovis,were observed as green fluorescent mycobacteria, whereas mycobacteria ofthe MOTT group, i.e. M. avium, M. intracellulare and M. kansasii, wereobserved as red fluorescent mycobacteria. Mycobacteria in the M.avium/M. tuberculosis mixture were identified by a mixture of both greenfluorescent mycobacteria and red fluorescent mycobacteria.

[0381] The results show that it is possible to distinguish betweendifferent Mycobacterium species in one smear using a mixture ofdifferently labelled probes. Such simultaneous detection andidentification of mycobacteria may further be extended to comprise threeor more differently labelled peptide nucleic acid probes.

Example 11

[0382] The ability of a peptide nucleic acid probes to hybridise toprecursor rRNA and further to distinguish between. precursor rRNA of M.tuberculosis and precursor rRNA of M. avium was investigated byfluorescence in situ hybridisation.

[0383] Smears were prepared as described in Example 5 and FISH werecarried out as described in Example 7 using a fluorescein-labelled probetargeting precursor rRNA of M. tuberculosis (OK 749). The results aregiven in Table 13. TABLE 13 OK 749 Mycobacterium 1000 nM M.tuberculosis + M. avium −

[0384] From the results, it can be concluded that it is possible todetect precursor rRNA, and further that is possible to distinguishbetween precursor rRNA from different mycobacterium species. Theapplication of peptide nucleic acid targeting precursor rRNA may beparticularly useful for measuring the mycobacterial growth and thus bean indicator of the viability of the mycobacteria. This would inparticular be important for monitoring of the effect of antibiotics inrelation to both treatment of tuberculosis and drug susceptibilitystudies.

Example 12

[0385] The ability of peptide nucleic acid probes for differentiation ofdrug susceptible and drug resistant mycobacteria was evaluated using afluorescein-labelled probe targeting the wild type sequence of 23S rRNAof M. avium and M. intracellulare together with rhodamine-labelledprobes targeting single point mutations associated with macrolideresistance in M. avium and M. intracellulare.

[0386] Smears were prepared as described in Example 5 from cultures ofM. avium (ATCC no. 25292) and M. intracellulare (ATCC no. 13950). Thesestrains are anticipated to contain the wild type sequence of rRNA.Macrolide resistant variants were not available. FISH was carried out asdescribed in Example 7 using a fluorescein-labelled peptide nucleic acidprobe targeting wild type 23S rRNA (OK 745) and a mixture ofrhodamine-labelled peptide nucleic acid probes targeting the threepossible mutations at position 2568 (OK 746) and at position 2569 (OK747) of M. avium 23S rDNA of GenBank entry X52917 (see FIG. 6). Theresults are given in Table 14. TABLE 14 OK 745/OK 746/OK 747Mycobacterium species 500/500/500 nM M. avium (wild type) +++(G)/−(R) M.intracellulare (wild type) +++(G)/−(R)

[0387] The results in Table 14 show that M. avium and M. intracellulareare detected with the fluorescein-labelled probe (OK 745) targeting M.avium and M. intracellulare wild types and not detected with the mixtureof rhodamine-labelled probes (OK 746 and OK 747) targeting single pointmutations associated with macrolide resistance. Such peptide nucleicacid probes targeting the wild type and drug resistant variants,respectively, may be important tools for both the prediction of anefficient therapy as well as for monitoring the effect of the treatment.

Example 13

[0388] To illustrate the speed with which peptide nucleic acid probespenetrate the mycobacterial cell wall and subsequently hybridise totheir target sequence the protocol described in Example 7 was modifiedto 15 minutes hybridisation time and the results compared with 90minutes hybridisation time. Smears were prepared as described in Example5. The results are given in Table 15. TABLE 15 OK 623 OK 689 25 nM 100nM 15 min 90 min 15 min 90 min M. tuberculosis ++ +++ M. avium ++ +++

[0389] The data presented in Table 15 show that hybridisation by peptidenucleic acid probes inside the mycobacterial cells is accomplished in avery short time resulting in a detectable signal after just 15 minutesincubation. Thus, the use peptide nucleic acid probes makes possible thedevelopment of very fast fluorescence in situ hybridisation protocols.

Example 14

[0390] To describe the ability of very short peptide nucleic acid probesto hybridise to target sequences, a 12-mer peptide nucleic acid probelabelled with fluorescein (OK 575) was tested by fluorescence in situhybridisation (FISH).

[0391] Smears were prepared as described in Example 5 and FISH werecarried out as described in Example 7. The results are given in Table16. TABLE 16 OK 575 Mycobacterium 50 nM M. tuberculosis + M. bovis BCG++ M. avium − M. intracellulare − M. kansasii −

[0392] The results in table 17 shows that a 12-mer peptide nucleic acidprobe is capable of hybridising specifically to target sequences underthe same stringency conditions as 15-mers. A lower florescence intensityis obtained as the T_(m) for a 12-mer peptide nucleic acid probe islower than T_(m) for a 1 5-mer peptide nucleic acid probe.

[0393] The data clearly suggest that by lowering the stringencycondition, e.g. by decreasing the hybridisation/washing temperatureand/or the concentration of formamide, even shorter probes may beapplied for detection of mycobacteria provided that specific sequencesof such can be designed.

1 123 15 basepairs nucleic acid basepairs single linear 1 AGATCGGGGTAGCAC 15 15 basepairs nucleic acid basepairs single linear 2 TGTTTTCTCCTCCTA 15 15 basepairs nucleic acid basepairs single linear 3 ACTGCCTCTCAGCCG 15 15 basepairs nucleic acid basepairs single linear 4 TGATACTAGGCAGGT 15 15 basepairs nucleic acid basepairs single linear 5 CGGATTCACAGCGGA 15 15 basepairs nucleic acid basepairs single linear 6 TCACCACCCTCCTCC 15 15 basepairs nucleic acid basepairs single linear 7 TTAACCTTGCGACAT 15 15 basepairs nucleic acid basepairs single linear 8 ACTATTCACACGCGC 15 15 basepairs nucleic acid basepairs single linear 9 CTCCGCGGTGAACCA 15 15 basepairs nucleic acid basepairs single linear 10 GCTTTACACCACGGC 15 15 basepairs nucleic acid basepairs single linear 11 ACGCTTGGGGGCCTT 15 15 basepairs nucleic acid basepairs single linear 12 CCACACCCACCACAA 15 15 basepairs nucleic acid basepairs single linear 13 CCGGTGGCTTCGCTG 15 15 basepairs nucleic acid basepairs single linear 14 ACTTGCCTTGTCGCT 15 15 basepairs nucleic acid basepairs single linear 15 GATTCGTCACGGGCG 15 15 basepairs nucleic acid basepairs single linear 16 AACTCCACACCCCCG 15 15 basepairs nucleic acid basepairs single linear 17 ACTCCACACCCCCGA 15 15 basepairs nucleic acid basepairs single linear 18 ACCCCTTCGCTTGAC 15 15 basepairs nucleic acid basepairs single linear 19 CTTGCCCCAGTGTTA 15 15 basepairs nucleic acid basepairs single linear 20 CTCTCCCTACCGGCT 15 15 basepairs nucleic acid basepairs single linear 21 GATATTCCGGTCCCC 15 15 basepairs nucleic acid basepairs single linear 22 ACTCCGCCCCAACTG 15 15 basepairs nucleic acid basepairs single linear 23 CTGTCCCTAAACCCG 15 15 basepairs nucleic acid basepairs single linear 24 TTCGAGGTTAGATGC 15 15 basepairs nucleic acid basepairs single linear 25 GTCCCTAAACCCGAT 15 15 basepairs nucleic acid basepairs single linear 26 GGTGCACCAGAGGTT 15 15 basepairs nucleic acid basepairs single linear 27 CTGGCGGGACAACTG 15 15 basepairs nucleic acid basepairs single linear 28 TTATCCTGACCGAAC 15 15 basepairs nucleic acid basepairs single linear 29 GACCTATTGAACCCG 15 15 basepairs nucleic acid basepairs single linear 30 GAAGAGACCTTTCCG 15 15 basepairs nucleic acid basepairs single linear 31 CACTCGAGTATCTCC 15 15 basepairs nucleic acid basepairs single linear 32 ATCACCCACGTGTTA 15 15 basepairs nucleic acid basepairs single linear 33 GCATCCCGTGGTCCT 15 15 basepairs nucleic acid basepairs single linear 34 CACAAGACATGCATC 15 15 basepairs nucleic acid basepairs single linear 35 TAAAGCGCTTTCCAC 15 15 basepairs nucleic acid basepairs single linear 36 GCTCATCCCACACCG 15 15 basepairs nucleic acid basepairs single linear 37 CCGAGAGAACCCGGA 15 15 basepairs nucleic acid basepairs single linear 38 AGTCCCCACCATTAC 15 15 basepairs nucleic acid basepairs single linear 39 AACCTCGCGGCATCG 15 15 basepairs nucleic acid basepairs single linear 40 GGCTTTTAAG GATTC 15 15 basepairs nucleic acid basepairs single linear 41GACCCCGATC CGAAC 15 15 basepairs nucleic acid basepairs single linear 42CCGACTTCAC GGGGT 15 15 basepairs nucleic acid basepairs single linear 43CGGAGGGGCA GTATC 15 15 basepairs nucleic acid basepairs single linear 44GATCAATGCT CGGTT 15 15 basepairs nucleic acid basepairs single linear 45TTCCCCGCGT TACCT 15 15 basepairs nucleic acid basepairs single linear 46TTAGCCTGTT CCGGT 15 15 basepairs nucleic acid basepairs single linear 47GCATGCGGTT TAGCC 15 15 basepairs nucleic acid basepairs single linear 48TACCCGGTTG TCCAT 15 15 basepairs nucleic acid basepairs single linear 49GTAGAGCTGA GACAT 15 15 basepairs nucleic acid basepairs single linear 50GCCGTCCCAG GCCAC 15 15 basepairs nucleic acid basepairs single linear 51CTCGGGTGTT GATAT 15 15 basepairs nucleic acid basepairs single linear 52ACTATTTCAC TCCCT 15 15 basepairs nucleic acid basepairs single linear 53ACGCCATCAC CCCAC 15 15 basepairs nucleic acid basepairs single linear 54CGACGTGTCC CTGAC 15 15 basepairs nucleic acid basepairs single linear 55ACTACACCCC AAAGG 15 15 basepairs nucleic acid basepairs single linear 56CACGCTTTTA CACCA 15 15 basepairs nucleic acid basepairs single linear 57GCGACTACAC ATCCT 15 15 basepairs nucleic acid basepairs single linear 58CGGCGCATAA TCACT 15 15 basepairs nucleic acid basepairs single linear 59CCACATCCAC CGTAA 15 15 basepairs nucleic acid basepairs single linear 60CGCTGAATGG GGGAC 15 15 basepairs nucleic acid basepairs single linear 61GGAGCTTCGC TGAAT 15 15 basepairs nucleic acid basepairs single linear 62CGGTCACCCG GAGCT 15 15 basepairs nucleic acid basepairs single linear 63GGACGCCCAT ACACG 15 15 basepairs nucleic acid basepairs single linear 64GAAGGGGAAT GGTCG 15 15 basepairs nucleic acid basepairs single linear 65AATCGCCACG CCCCC 15 15 basepairs nucleic acid basepairs single linear 66CAGCGAAGGT CCCAC 15 15 basepairs nucleic acid basepairs single linear 67GTCACCCCAT TGCTT 15 15 basepairs nucleic acid basepairs single linear 68ATCGCTCTCT ACGGG 15 15 basepairs nucleic acid basepairs single linear 69GTGTATGTGC TCGCT 15 15 basepairs nucleic acid basepairs single linear 70ACGGTATTCC GGGCC 15 15 basepairs nucleic acid basepairs single linear 71GGCCGAATCC CGCTC 15 15 basepairs nucleic acid basepairs single linear 72AAACAGTCGC TACCC 15 15 basepairs nucleic acid basepairs single linear 73CCTTACGGGT TAACG 15 15 basepairs nucleic acid basepairs single linear 74GAGACAGTTG GGAAG 15 15 basepairs nucleic acid basepairs single linear 75TGGCGTCTGT GCTTC 15 15 basepairs nucleic acid basepairs single linear 76CGACTCCACA CAAAC 15 15 basepairs nucleic acid basepairs single linear 77GATAAGGGTT CGACG 15 15 basepairs nucleic acid basepairs single linear 78ATCCGTTGAG TGACA 15 15 basepairs nucleic acid basepairs single linear 79CAGCCCGTTA TCCCC 15 15 basepairs nucleic acid basepairs single linear 80AACCTTTGGG ACCTG 15 15 basepairs nucleic acid basepairs single linear 81TAAAAGGGTG AGAAA 15 15 basepairs nucleic acid basepairs single linear 82GTCTGGCCTA TCAAT 15 15 basepairs nucleic acid basepairs single linear 83AGATTGCCCA CGTGT 15 15 basepairs nucleic acid basepairs single linear 84AATCCGAGAA AACCC 15 15 basepairs nucleic acid basepairs single linear 85GCATTACCCG CTGGC 15 15 basepairs nucleic acid basepairs single linear 86TTAAAAGGAT TCGCT 15 15 basepairs nucleic acid basepairs single linear 87AGACCCCAAT CCGAA 15 15 basepairs nucleic acid basepairs single linear 88GACTCCGACT TCATG 15 15 basepairs nucleic acid basepairs single linear 89GTCTTTTCGT CCTGC 15 15 basepairs nucleic acid basepairs single linear 90GTCTTATCGT CCTGC 15 15 basepairs nucleic acid basepairs single linear 91GTCTTCTCGT CCTGC 15 15 basepairs nucleic acid basepairs single linear 92GTCTTGTCGT CCTGC 15 15 basepairs nucleic acid basepairs single linear 93GTCTATTCGT CCTGC 15 15 basepairs nucleic acid basepairs single linear 94GTCTCTTCGT CCTGC 15 15 basepairs nucleic acid basepairs single linear 95GTCTGTTCGT CCTGC 15 15 basepairs nucleic acid basepairs single linear 96TTGGCCGGTG CTTCT 15 15 basepairs nucleic acid basepairs single linear 97TTGGCCGGTA CTTCT 15 15 basepairs nucleic acid basepairs single linear 98TTGGCCGGTC CTTCT 15 15 basepairs nucleic acid basepairs single linear 99TTGGCCGGTT CTTCT 15 15 basepairs nucleic acid basepairs single linear100 ACCGCGGCTG CTGGC 15 15 basepairs nucleic acid basepairs singlelinear 101 ACCGCGGCTA CTGGC 15 15 basepairs nucleic acid basepairssingle linear 102 ACCGCGGCTC CTGGC 15 15 basepairs nucleic acidbasepairs single linear 103 ACCGCGGCTT CTGGC 15 15 basepairs nucleicacid basepairs single linear 104 CGGCAGCTGG CACGT 15 15 basepairsnucleic acid basepairs single linear 105 CGGCCGCTGG CACGT 15 15basepairs nucleic acid basepairs single linear 106 CGGCTGCTGG CACGT 1515 basepairs nucleic acid basepairs single linear 107 CGTATTACCG CAGCT15 15 basepairs nucleic acid basepairs single linear 108 CGTATTACCGCCGCT 15 15 basepairs nucleic acid basepairs single linear 109CGTATTACCG CTGCT 15 15 basepairs nucleic acid basepairs single linear110 TTCCTTTGAG TTTTA 15 15 basepairs nucleic acid basepairs singlelinear 111 TTCCTTTAAG TTTTA 15 15 basepairs nucleic acid basepairssingle linear 112 TTCCTTTCAG TTTTA 15 15 basepairs nucleic acidbasepairs single linear 113 TTCCTTTTAG TTTTA 15 15 basepairs nucleicacid basepairs single linear 114 TTCCTTAGAG TTTTA 15 15 basepairsnucleic acid basepairs single linear 115 TTCCTTCGAG TTTTA 15 15basepairs nucleic acid basepairs single linear 116 TTCCTTGGAG TTTTA 1515 basepairs nucleic acid basepairs single linear 117 CATGTGTCCT GTGGT15 15 basepairs nucleic acid basepairs single linear 118 CGTCAGCCCGAGAAA 15 15 basepairs nucleic acid basepairs single linear 119CACTACACAC GCTCG 15 15 basepairs nucleic acid basepairs single linear120 TGGCGTTGAG GTTTC 15 15 basepairs nucleic acid basepairs singlelinear 121 GTCCGTGAAC CCGAT 15 15 basepairs nucleic acid basepairssingle linear 122 TACGCTCTTT GAGCT 15 15 basepairs nucleic acidbasepairs single linear 123 AACACTCCCT TTGGA 15

We claim
 1. Peptide nucleic acid probe for detecting a target sequenceof one or more mycobacteria optionally present in a sample, said probebeing capable of hybridising to a target sequence of mycobacterial rDNA,precursor rRNA or rRNA forming detectable hybrids, and a mixture of suchprobes.
 2. Peptide nucleic acid probe according to claim 1, said probebeing capable of hybridising to a target sequence of mycobacterial rDNA,precursor rRNA, or 23S, 16S or 5S rRNA forming detectable hybrids, and amixture of such probes.
 3. Peptide nucleic acid probe according to claim1, said probe being capable of hybridising to a target sequence ofmycobacterial rDNA, precursor rRNA, or 23S, 16S or 5S rRNA formingdetectable hybrids, said target sequence being obtainable by (a)comparing the nucleobase sequences of said mycobacterial rRNA or rDNA ofone or more mycobacteria to be detected with the correspondingnucleobase sequence of organism(s), in particular other mycobacteria, inparticular other mycobacteria, from which said one or more mycobacteriaare to be distinguished, (b) selecting a target sequence of said rRNA orrDNA which includes at least one nucleobase differing from thecorresponding nucleobase of the organism(s), in particular othermycobacteria, from which said one or more mycobacteria are to bedistinguished, and (c) determining the capability of said probe tohybridise to the selected target sequence to form detectable hybrids,and a mixture of such probes.
 4. Peptide nucleic acid probe according toclaim 1, said probe being capable of hybridising to a target sequence ofmycobacterial rDNA, precursor rRNA or 23S, 16S or 5S rRNA formingdetectable hybrids, said probe being obtainable by (a) comparing thenucleobase sequences of said mycobacterial rRNA or rDNA of one or moremycobacteria to be detected with the corresponding nucleobase sequenceof organism(s), in particular other mycobacteria, in particular othermycobacteria, from which said one or more mycobacteria are to bedistinguished, (b) selecting a target sequence of said rRNA or rDNAwhich includes at least one nucleobase differing from the correspondingnucleobase of the organism(s), in particular other mycobacteria, fromwhich said one or more mycobacteria are to be distinguished, (c)synthesising said probe, and (d) determining the capability of saidprobe to hybridise to the selected target sequence to form detectablehybrids, and a mixture of such probes.
 5. Peptide nucleic acid probeaccording to claim 1 for detecting a target sequence of one or moremycobacteria of the Mycobacterium tuberculosis Complex (MTC) or fordetecting a target sequence of one or more mycobacteria other thanmycobacteria of the Mycobacterium tuberculosis Complex (MOTT) optionallypresent in a sample, which probe comprises from 6 to 30 polymerisedpeptide nucleic acid moieties, said probe being capable of hybridisingto a target sequence of mycobacterial rDNA, precursor rRNA or 23S, 16Sor 5S rRNA forming detectable hybrids, and a mixture of such probes. 6.Peptide nucleic acid probe according to claim 1 for detecting a targetsequence of rDNA, precursor rRNA or 23S, 16S or 5S rRNA of one or moremycobacteria of the Mycobacterium tuberculosis Complex (MTC) or fordetecting a target sequence of rDNA, precursor rRNA or 23S, 16S or 5SrRNA of one or more mycobacteria other than mycobacteria of theMycobacterium tuberculosis Complex (MOTT) optionally present in asample, which probe comprises from 10 to 30 polymerised moieties offormula (I)

wherein each X and Y independently designate O or S, each Zindependently designates O, S, NR¹, or C(R¹)₂, wherein each R¹independently designate H, C₁₋₆ alkyl, C₁₋₆ alkenyl, C₁₋₆ alkynyl, eachR², R³ and R⁴ designate independently H, the side chain of a naturallyoccurring amino acid, the side chain of a non-naturally occurring aminoacid, C₁₋₄ alkyl, C₁₋₄ alkenyl or C₁₋₄ alkynyl, or a functional group,each Q independently designates a naturally occurring nucleobase, anon-naturally occurring nucleobase, an intercalator, anucleobase-binding group, a label or H, with the proviso that the probecomprising such subsequence is capable of forming detectable hybridswith the target sequence of said mycobacterial rDNA, precursor rRNA or23S, 16S or 5S rRNA, and a mixture of such probes.
 7. Peptide nucleicacid probe according to claim 1 for detecting a target sequence of 23SrRNA of one or more mycobacteria of the Mycobacterium tuberculosisComplex (MTC) optionally present in a sample, which probe comprises from10 to 30 polymerised moieties of formula (I) as defined in claim 6, withthe proviso that the Qs of adjacent moieties are selected so as to forma sequence of which a subsequence includes at least one nucleobase thatis complementary to a nucleobase of M. tuberculosis 23S rRNA differingfrom the corresponding nucleobase of at least M. avium located withinthe following domains Positions 149-158 in FIG. 1A, Positions 220-221 inFIG. 1A, Positions 328-361 in FIG. 1A and FIG. 1B, Positions 453-455 inFIG. 1B. Positions 490-501 in FIG. 1B, Positions 637-660 in FIG. 1C,Positions 706-712 in FIG. 1D, Positions 762-789 in FIG. 1D, Position 989in FIG. 1D, Positions 1068-1072 in FIG. 1D, Position 1148 in FIG. 1E,Positions 1311-1329 in FIG. 1E, Positions 1361-1364 in FIG. 1F, Position1418 in FIG. 1F, Positions 1563-1570 in FIG. 1F, Positions 1627-1638 inFIG. 1G, Positions 1675-1677 in FIG. 1G, Position 1718 in FIG. 1G,Positions 1734-1740 in FIG. 1H, Positions 1967-1976 in FIG. 1H,Positions 2403-2420 in FIG. 1H, Positions 2457-2488 in FIG. 1I,Positions 2952-2956 in FIG. 1I, Positions 2966-2969 in FIG. 1J,Positions 3000-3003 in FIG. 1J or Positions 3097-3106 in FIG. 1J, andfurther with the proviso that the probe comprising such subsequence iscapable of forming detectable hybrids with a target sequence of saidmycobacterial 23S rRNA, and a mixture of such probes.
 8. Peptide nucleicacid probe according to claim 1 for detecting a target sequence of 16SrRNA of one or more mycobacteria of the Mycobacterium tuberculosisComplex (MTC) optionally present in a sample, which probe comprises from10 to 30 polymerised moieties of formula (I) as defined in claim 6, withthe proviso that the Qs of adjacent moieties are selected so as to forma sequence of which a subsequence includes at least one nucleobase thatis complementary to a nucleobase of M. tuberculosis 16S rRNA differingfrom the corresponding nucleobase of at least M. avium located withinthe following domains Positions 76-79 in FIG. 2A, Positions 98-101 inFIG. 2A, Positions 135-136 in FIG. 2A, Positions 194-201 in FIG. 2B,Positions 222-229 in FIG. 2B, Position 242 in FIG. 2B, Position 474 inFIG. 2C, Positions 1136-1145 in FIG. 2C, Positions 1271-1272 in FIG. 2C,Positions 1287-1292 in FIG. 2D, Position 1313 in FIG. 2D, or Position1334 in FIG. 2D, and further with the proviso that the probe comprisingsuch subsequence is capable of forming detectable hybrids with a targetsequence of said mycobacterial 16S rRNA, and a mixture of such probes.9. Peptide nucleic acid probe according to claim 1 for detecting atarget sequence of 5S rRNA of one or more mycobacteria of theMycobacterium tuberculosis Complex (MTC) optionally present in a sample,which probe comprises from 10 to 30 polymerised moieties of formula (I)as defined in claim 6, with the proviso that the Qs of adjacent moietiesare selected so as to form a sequence of which a subsequence includes atleast one nucleobase that is complementary to a nucleobase of M.tuberculosis 5S rRNA differing from the corresponding nucleobase of atleast M. avium located within the following domain Positions 86-90 inFIG. 3 and further with the proviso that the probe comprising suchsubsequence is capable of forming detectable hybrids with a targetsequence of said mycobacterial 5S rRNA, and a mixture of such probes.10. Peptide nucleic acid probe according to claim 7 or 8 for detecting atarget sequence of 23S or 16S rRNA of one or more mycobacteria of theMycobacterium tuberculosis Complex (MTC) optionally present in a sample,which probe comprises from 10 to 30 polymerised moieties of formula (I)as defined in claim 6, with the proviso that the Qs of adjacent moietiesare selected so as to form a sequence of which a subsequence includes atleast one nucleobase that is complementary to a nucleobase of M.tuberculosis 23S or 16 S rRNA differing from the correspondingnucleobase of at least M. avium located within the following domainsPositions 149-158 in FIG. 1A, Positions 328-361 in FIG. 1A and FIG. 1B,Positions 490-501 in FIG. 1B, Positions 637-660 in FIG. 1C, Positions762-789 in FIG. 1D, Positions 1068-1072 in FIG. 1D, Positions 1311-1329in FIG. 1E, Positions 1361-1364 in FIG. 1F, Positions 1563-1570 in FIG.1F, Positions 1627-1638 in FIG. 1G, Positions 1734-1740 in FIG. 1H,Positions 2457-248B in FIG. 1I, Positions 2952-2956 in FIG. 1I,Positions 3097-3106 in FIG. 1J, Positions 135-136 in FIG. 2A, orPositions 1287-1292 in FIG. 2D, and further with the proviso that theprobe comprising such subsequence is capable of forming detectablehybrids with a target sequence of said mycobacterial 23S or 16S rRNA,and a mixture of such probes.
 11. Peptide nucleic acid probe accordingto claim 1 for detecting a target sequence of 23S rRNA of one or moremycobacteria other than mycobacteria of the Mycobacterium tuberculosisComplex (MOTT) optionally present in a sample, which probe comprisesfrom 10 to 30 polymerised moieties of formula (I) as defined in claim 6,with the proviso that the Qs of adjacent moieties are selected so as toform a sequence of which a subsequence includes at least one nucleobasethat is complementary to a nucleobase of M. avium 23S rRNA differingfrom the corresponding nucleobase of at least M. tuberculosis locatedwithin the following domains Positions 99-101 in FIG. 4A, Position 183in FIG. 4A, Positions 261-271 in FIG. 4A, Positions 281-284 in FIG. 4B,Positions 290-293 in FIG. 4B, Positions 327-335 in FIG. 4B, Positions343-357 in FIG. 4B, Positions 400-405 in FIG. 4B and FIG. 4C, Positions453-462 in FIG. 4C, Positions 587-599 in FIG. 4C, Positions 637-660 inFIG. 4D, Positions 704-712 in FIG. 4D, Positions 763-789 in FIG. 4E,Positions 1060-1074 in FIG. 4E, Positions 1177-1185 in FIG. 4E,Positions 1259-1265 in FIG. 4F, Positions 1311-1327 in FIG. 4F,Positions 1345-1348 in FIG. 4F, Positions 1361-1364 in FIG. 4G,Positions 1556-1570 in FIG. 4G, Positions 1608-1613 in FIG. 4H,Positions 1626-1638 in FIG. 4H, Positions 1651-1659 in FIG. 4H,Positions 1675-1677 in FIG. 4H, Positions 1734-1741 in FIG. 4H,Positions 1847-1853 in FIG. 4I, Positions 1967-1976 in FIG. 4I,Positions 2006-2010 in FIG. 4I, Positions 2025-2027 in FIG. 4I,Positions 2131-2132 in FIG. 4J, Positions 2252-2255 in FIG. 4J,Positions 2396-2405 in FIG. 4J and FIG. 4K, Positions 2416-2420 in FIG.4K, Positions 2474-2478 in FIG. 4K, Position 2687 in FIG. 4K, Position2719 in FIG. 4K, Position 2809 in FIG. 4L, Positions 3062-2068 in FIG.4L, or Positions 3097-3106 in FIG. 4L, and further with the proviso thatthe probe comprising such subsequence is capable of forming detectablehybrids with a target sequence of said mycobacterial 23S rRNA, and amixture of such probes.
 12. Peptide nucleic acid probe according toclaim 1 for detecting a target sequence of 16S rRNA of one or moremycobacteria other than mycobacteria of the Mycobacterium tuberculosisComplex (MOTT) optionally present in a sample, which probe comprisesfrom 10 to 30 polymerised moieties of formula (I) as defined in claim 6,with the proviso that the Qs of adjacent moieties are selected so as toform a sequence of which a subsequence includes at least one nucleobasethat is complementary to a nucleobase of M. avium 16S rRNA differingfrom the corresponding nucleobase of at least M. tuberculosis locatedwithin the following domains Positions 135-136 in FIG. 5A, Positions472-475 in FIG. 5A. Positions 1136-1144 in FIG. 5A, Positions 1287-1292in FIG. 5B, Position 1313 in FIG. 5B, or Position 1334 in FIG. 5B, andfurther with the proviso that the probe comprising such subsequence iscapable of forming detectable hybrids with a target sequence of saidmycobacterial 16S rRNA, and a mixture of such probes.
 13. Peptidenucleic acid probe according to claim 11 or 12 for detecting a targetsequence of 23S or 16S rRNA of one or more mycobacteria other thanmycobacteria of the Mycobacterium tuberculosis Complex (MOTT) optionallypresent in a sample, which probe comprises from 10 to 30 polymerisedmoieties of formula (I) as defined in claim 6, with the proviso that theQs of adjacent moieties are selected so as to form a sequence of which asubsequence includes at least one nucleobase that is complementary to anucleobase of M. avium 23S or 16S rRNA differing from the correspondingnucleobase of at least M. tuberculosis located within the followingdomains Positions 99-101 in FIG. 4A, Positions 290-293 in FIG. 4B,Positions 400-405 in FIG. 4B and FIG. 4C, Positions 453-462 in FIG. 4C,Positions 637-660 in FIG. 4D, Positions 763-789 in FIG. 4E, Positions1311-1327 in FIG. 4F, Positions 1361-1364 in FIG. 4G, Positions1734-1741 in FIG. 4H, Positions 2025-2027 in FIG. 4I, Positions2474-2478 in FIG. 4K, Positions 3062-2068 in FIG. 4L, or Positions1287-1292 in FIG. 5B, and further with the proviso that the probecomprising such subsequence is capable of forming detectable hybridswith a target sequence of said mycobacterial 23S or 16S rRNA, and amixture of such probes.
 14. Peptide nucleic acid probe according toclaim 1 for detecting a target sequence of 23S, 16S or 5S rRNA of one ormore mycobacteria of the Mycobacterium tuberculosis Complex (MTC) or fordetecting a target sequence of 23S, 16S or 5S rRNA of one or moremycobacteria other than mycobacteria of the Mycobacterium tuberculosisComplex (MOTT) optionally present in a sample, which probe comprisesfrom 10 to 30 polymerised moieties of formula (I) as defined in claim 6,with the proviso that the Qs of adjacent moieties are selected so as toform a sequence of which a subsequence includes at least one nucleobasethat is complementary to a nucleobase that differs from thecorresponding nucleobase of 23S, 16S or 5S rRNA of said one or moremycobacteria located within the following domains positions 2568-2569 inFIG. 6, Position 452 in FIG. 7, Positions 473-477 in FIG. 7, orPositions 865-866 in FIG. 7, and further with the proviso that the probecomprising such subsequence is capable of forming detectable hybridswith the target sequence of said mycobacterial 23S, 16S or 5S rRNA, anda mixture of such probes.
 15. Peptide nucleic acid probe according toclaim 6 of formula (II), (III), or (IV)

wherein Z, R², R³, and R⁴, and Q is as defined in claim 6 with theprovisos defined in claims 6 to 14, and a mixture of such probes. 16.Peptide nucleic acid probe according to claim 6, wherein Z is NH, NCH₃or O, each R², R³ and R⁴ independently designate H or the side chain ofa naturally occurring amino acid, the side chain of a non-naturallyoccurring amino acid, or C₁₋₄ alkyl, and each Q is a naturally occurringnucleobase or a non-naturally occurring nucleobase with the provisosdefined in claims 6 to 14, and a mixture of such probes.
 17. Peptidenucleic acid probe according to claim 6, wherein Z is NH or O, and R² isH or the side chain of Ala, Asp, Cys, Glu, His, HomoCys, Lys, Orn, Seror Thr, and 0 is a nucleobase selected from thymine, adenine, cytosine,guanine, uracil, iso-C and 2,6-diaminopurine with the provisos definedin claims 6 to 14, and a mixture of such probes.
 18. Peptide nucleicacid probe according to claim 6 of formula (V)

wherein R⁴ is H or the side chain of Ala, Asp, Cys. Glu, His, HomoCys,Lys, Orn, Ser or Thr, and Q is as defined in claim 17 with the provisosdefined in claims 6 to 14, and a mixture of such probes.
 19. Peptidenucleic acid probe according to claim 1 further comprising one or morelabels and a mixture of such probes, which labels may be mutuallyidentical or different, which probes optionally may comprise one or morelinkers. and which probes may be mutually identical or different withthe provisos defined in claims 6 to
 14. 20. Peptide nucleic acid probeaccording to claim 1 for detecting a target sequence of one or moremycobacteria, the nucleobase sequence of said probe being substantiallycomplementary to the nucleobase sequence of said target sequence. 21.Peptide nucleic acid probe according to claim 1 for detecting a targetsequence of one or more mycobacteria, the nucleobase sequence of saidprobe being complementary to the nucleobase sequence of said targetsequence.
 22. Peptide nucleic acid probes according to claim 6 whereinthe Qs of adjacent moieties are selected so as to form the followingsubsequences AGA TGC GGG TAG CAC (selected from positions 149-158 inFIG. 1A), (Seq ID no 1) TGT TTT CTC CTC CTA (selected from positions220-221 in FIG. 1A), (Seq ID no 2) ACT GCC TCT CAG CCG (selected frompositions 328-361 in FIG. 1A and FIG. 1B), (Seq ID no 3) TGA TAC TAG GCAGGT (selected from positions 453-455 in FIG. 1B), (Seq ID no 4) CGG ATTCAC AGC GGA (selected from positions 490-501 in FIG. 1B), (Seq ID no 5)TCA CCA CCC TCC TCC (selected from positions 637-660 in FIG. 1C), (SeqID no 6) CCA CCC TCC TCC (selected from positions 637-660 in FIG. 1C),(modified Seq ID no 6) TTA ACC TTG CGA CAT (selected from positions706-712 in FIG. 1C), (Seq ID no 7) ACT ATT CAC ACG CGC (selected frompositions 762-789 in FIG. 1D), (Seq ID no 8) CTC CGC GGT GAA CCA(selected from position 989 in FIG. 1D), (Seq ID no 9) GCT TTA CAC CACGGC (selected from positions 1068-1072 in FIG. 1D), (Seq ID no 10) ACGCTT GGG GGC CTT (selected from position 1148 in FIG. 1E), (Seq ID no 11)CCA CAC CCA CCA CAA (selected from positions 1311-1329 in FIG. 1F), (SeqID no 12) CCG GTG GCT TCG CTG (selected from positions 1361-1364 in FIG.1F), (Seq ID no 13) ACT TGC CTT GTC GCT (selected from position 1418 inFIG. 1F), (Seq ID no 14) GAT TCG TCA CGG GCG (selected from positions1563-1570 in FIG. 1F), (Seq ID no 15) AAC TCC ACA CCC CCG (selected frompositions 1627-1638 in FIG. 1G), (Seq ID no 16) ACT CCA CAC CCC CGA(selected from positions 1627-1638 in FIG. 1G), (Seq ID no 17) ACC CCTTCG CTT GAC (selected from positions 1675-1677 in FIG. 1G), (Seq ID no18) CTT GCC CCA GTG TTA (selected from position 1718 in FIG. 1G), (SeqID no 19) CTC TCC CTA CCG GCT (selected from positions 1734-1740 in FIG.1H), (Seq ID no 20) GAT ATT CCG GTC CCC (selected from positions1967-1976 in FIG. 1H), (Seq ID no 21) ACT CCG CCC CAA CTG (selected frompositions 2403-2420 in FIG. 1H), (Seq ID no 22) CTG TCC CTA AAC CCG(selected from positions 2457-2488 in FIG. 1I), (Seq ID no 23) TTC GAGGTT AGA TGC (selected from positions 2457-2488 in FIG. 1I), (Seq ID no24) GTC CCT AAA CCC GAT (selected from positions 2457-2486 in FIG. 1I),(Seq ID no 25) GGT GCA CCA GAG GTT (selected from positions 2952-2956 inFIG. 1I), (Seq ID no 26) CTG GCG GGA CAA CTG (selected from positions2966-2969 in FIG. 1J), (Seq ID no 27) TTA TCC TGA CCG AAC (selected frompositions 3000-3003 in FIG. 1J), (Seq ID no 28) GAC CTA TTG AAC CCG(selected from positions 3097-3106 in FIG. 1J), (Seq ID no 29) GAA GAGACC TTT CCG (selected from positions 76-79 in FIG. 2A), (Seq ID no 30)CAC TCG AGT ATC TCC (selected from positions 98-101 in FIG. 2A), (Seq IDno 31) ATC ACC CAC GTG TTA (selected from positions 136-136 in FIG. 2A),(Seq ID no 32) GCA TCC CGT GGT CCT (selected from positions 194-201 inFIG. 2B), (Seq ID no 33) CAC AAG ACA TGC ATC (selected from positions194-201 in FIG. 2B), (Seq ID no 34) TAA AGC GCT TTC CAC (selected frompositions 222-229 in FIG. 2B), (Seq ID no 35) GCT CAT CCC ACA CCG(selected from position 242 in FIG. 2B), (Seq ID no 36) CCG AGA GAA CCCGGA (selected from position 474 in FIG. 2C), (Seq ID no 37) AGT CCC CACCAT TAC (selected from positions 1136-1146 in FIG. 2C), (Seq ID no 38)AAC CTC GCG GCA TCG (selected from positions 1271-1272 in FIG. 2C), (SeqID no 39) GGC TTT TAA GGA TTC (selected from positions 1287-1292 in FIG.2D), (Seq ID no 40) GAC CCC GAT CCG AAC (selected from position 1313 inFIG. 2D), (Seq ID no 41) CCG ACT TCA CGG GGT (selected from position1334 in FIG. 2D), (Seq ID no 42) CGG AGG GGC AGT ATC (selected frompositions 86-90 in FIG. 3), (Seq ID no 43) GAT CAA TGC TCG GTT (selectedfrom positions 99-101 in FIG. 4A), (Seq ID no 44) TTC CCC GCG TTA CCT(selected from position 183 in FIG. 4A), (Seq ID no 45) TTA GCC TGT TCCGGT (selected trom positions 261-271 in FIG. 4A), (Seq ID no 46) GCA TGCGGT TTA GCC (selected from positions 281-284 in FIG. 4B), (Seq ID no 47)TAC CCG GTT GTC CAT (selected from positions 290-293 in FIG. 4B), (SeqID no 48) GTA GAG CTG AGA CAT (selected from positions 327-335 and343-357 in FIG. 4B), (Seq ID no 49) GCC GTC CCA GGC CAC (selected frompositions 400-405 in FIG. 48 and FIG. 4C), (Seq ID no 50) CTC GGG TGTTGA TAT (selected from positions 453-462 in FIG. 4C), (Seq ID no 51) ACTATT TCA CTC CCT (selected from positions 587-599 in FIG. 4D), (Seq ID no52) ACG CCA TCA CCC CAC (selected from positions 637-660 in FIG. 4D),(Seq ID no 53) CGA CGT GTC CCT CAC (selected from positions 704-712 inFIG. 4D), (Seq ID no 54) ACT ACA CCC CAA AGG (selected from positions763-789 in FIG. 4E), (Seq ID no 55) CAC GCT TTT ACA CCA (selected frompositions 1060-1074 in FIG. 4E), (Seq ID no 56) GCG ACT ACA CAT CCT(selected from positions 1177-1185 in FIG. 4E), (Seq ID no 57) CGG CCCATA ATC ACT (selected from positions 1259-1265 in FIG. 4F), (Seq ID no58) CCA CAT CCA CCC TAA (selected from positions 1311-1327 in FIG. 4F),(Seq ID no 59) CGC TGA ATG GGG CAC (selected from positions 1345-1348 inFIG. 4F), (Seq ID no 60) GGA GCT TCG CTG AAT (selected from positions1361-1364 in FIG. 4G), (Seq ID no 61) CGG TCA CCC GGA GCT (selected frompositions 1361-1364 in FIG. 4G), (Seq ID no 62) GGA CCC CCA TAC ACG(selected from positions 1556-1570 in FIG. 4G), (Seq ID no 63) GAA GGGGAA TGG TCG (selected from positions 1608-1613 in FIG. 4H), (Seq ID no64) AAT CGC CAC CCC CCC (selected from positions 1626-1638 in FIG. 4H),(Seq ID no 65) CAG CGA AGG TCC CAC (selected from positions 1651-1659 inFIG. 4H), (Seq ID no 66) GTC ACC CCA TTG CTT (selected from positions1675-1677 in FIG. 4H), (Seq ID no 67) ATC GCT CTC TAC GGG (selected frompositions 1734-1741 in FIG. 4H), (Seq ID no 68) GTG TAT GTG CTC GCT(selected from positions 1847-1853 in FIG. 4I), (Seq ID no 69) ACG GTATTC CGG GCC (selected from positions 1967-1976 in FIG. 4I), (Seq ID no70) GGC CGA ATC CCG CTC (selected from positions 2006-2010 in FIG. 4I),(Seq ID no 71) AAA CAC TCG CTA CCC (selected from positions 2025-2027 inFIG. 4I), (Seq ID no 72) CCT TAC GGG TTA ACG (selected from positions2131-2132 in FIG. 4J), (Seq ID no 73) GAG ACA GTT GGG AAG (selected frompositions 2252-2255 in FIG. 4J), (Seq ID no 74) TGG CGT CTG TGC TTC(selected from positions 2396-2405 in FIG. 4J and FIG. 4K), (Seq ID no75) CGA CTC CAC ACA AAC (selected from positions 2416-2420 in FIG. 4K),(Seq ID no 76) GAT AAG GGT TCG ACG (selected from positions 2474-2478 inFIG. 4K), (Seq ID no 77) ATC CGT TGA GTG ACA (selected from position2687 in FIG. 4K), (Seq ID no 78) GAG CCC GTT ATC CCC (selected fromposition 2719 in FIG. 4K), (Seq ID no 79) AAC CTT TGG GAG CTG (selectedfrom position 2809 in FIG. 4L), (Seq ID no 80) TAA AAG GGT GAG AAA(selected from positions 3062-3068 in FIG. 4L), (Seq ID no 81) GTC TGGCCT ATC AAT (selected from positions 3097-3106 in FIG. 4L), (Seq ID no82) AGA TTG CCC ACG TGT (selected from positions 135-136 in FIG. 5A),(Seq ID no 83) AAT CCG AGA AAA CCC (selected from positions 427-475 inFIG. 5A), (Seq ID no 84) GCA TTA CCC GCT GGC (selected from positions1136-1144 in FIG. 5B), (Seq ID no 85) TTA AAA GGA TTC GCT (selected frompositions 1287-1292 in FIG. 5B), (Seq ID no 86) AGA CCC CAA TCC GAA(selected from position 1313 in FIG. 5B), (Seq ID no 87) GAC TCC GAC TTCATG (selected from positions 1334 in FIG. 5B), (Seq ID no 88) GTC TTTTCG TCC TGC (selected from positions 2568-2569 in FIG. 6), (Seq ID no89) GTC TTA TCG TCC TGC (selected from positions 2568 in FIG. 6), (SeqID no 90) GTC TTC TCG TCC TGC (selected from positions 2568 in FIG. 6),(Seq ID no 91) GTC TTG TCG TCC TGC (selected from positions 2568 in FIG.6), (Seq ID no 92) GTC TAT TCG TCC TGC (selected from positions 2568 inFIG. 6), (Seq ID no 93) GTC TCT TCG TCC TGC (selected from positions2568 in FIG. 6), (Seq ID no 94) GTC TGT TCG TCC TGC (selected frompositions 2568 in FIG. 6), (Seq ID no 95) TTG GCC GGT GCT TCT (selectedfrom position 452 in FIG. 7), (Seq ID no 96) TTG GCC GGT ACT TCT(selected from position 452 in FIG. 7), (Seq ID no 97) TTG GCC GGT CCTTCT (selected from position 452 in FIG. 7), (Seq ID no 98) TTG GCC GGTTCT TCT (selected from position 452 in FIG. 7), (Seq ID no 99) ACC GCGGCT GCT GGC (selected from positions 473-477 in FIG. 7), (Seq ID no 100)ACC GCG GCT ACT GGC (selected from positions 473 in FIG. 7), (Seq ID no101) ACC GCG GCT CCT GGC (selected from positions 473 in FIG. 7), (SeqID no 102) ACC GCG GCT TCT GGC (selected from positions 473 in FIG. 7),(Seq ID no 103) CGG CAG CTG GCA CGT (selected from positions 474 in FIG.7), (Seq ID no 104) CGG CCG CTG GCA CGT (selected from positions 474 inFIG. 7), (Seq ID no 105) CGG CTG CTG GCA CGT (selected from positions474 in FIG. 7), (Seq ID no 106) CGT ATT ACC GCA GCT (selected frompositions 477 in FIG. 7), (Seq ID no 107) CGT ATT ACC GCC GCT (selectedfrom positions 477 in FIG. 7), (Seq ID no 107) CGT ATT ACC GCT GCT(selected from positions 477 in FIG. 7), (Seq ID no 109) TTC CTT TGA GTTTTA (selected from positions 865-866 in FIG. 7), (Seq ID no 110) TTC CTTTAA GTT TTA (selected from positions 865 in FIG. 7), (Seq ID no 111) TTCCTT TCA GTT TTA (selected from positions 865 in FIG. 7), (Seq ID no 112)TTC CTT TTA GTT TTA (selected from positions 865 in FIG. 7), (Seq ID no113) TTC CTT AGA GTT TTA (selected from positions 865 in FIG. 7), (SeqID no 114) TTC CTT CGA GTT TTA (selected from positions 866 in FIG. 7),(Seq ID no 115) TTC GTT GGA GTT TTA (selected from positions 866 in FIG.7), (Seq ID no 116) CAT GTG TGC TGT GGT (Seq ID no 117) CGT GAG CCC GAGAAA (Seq ID no 118) GAG TAG AGA CGG TGG (Seq ID no 119) TGG CGT TGA GGTTTG and (Seq ID no 120) AAC ACT CCC TTT GGA (Seq ID no 123)

and a mixture of such probes.
 23. Peptide nucleic acid probes accordingto claim 22, wherein the Qs of adjacent moieties are selected so as toform the following subsequences TCA CCA CCC TCC TCC (Seq ID no 6) CCACCC TCC TCC (modified Seq ID no 6) ACT ATT CAC ACG CGC (Seq ID no 8) CCACAC CCA CCA CAA (Seq ID no 12) AAC TCC ACA CCC CCG (Seq ID no 16) ACTCCA CAC CCC CGA (Seq ID no 17) ACT CCG CCC CAA CTG (Seq ID no 22) CTGTCC CTA AAC CCG (Seq ID no 23) TTC GAG GTT AGA TGC (Seq ID no 24) GTCCCT AAA CCC GAT (Seq ID no 25) GAC CTA TTG AAC CCG (Seq ID no 29) GCATCC CGT GGT CCT (Seq ID no 33) CAC AAG ACA TGC ATC (Seq ID no 34) GGCTTT TAA GGA TTC (Seq ID no 40) GAT CAA TGC TCG GTT (Seq ID no 44) CGACTC CAC ACA AAC (Seq ID no 76) GCA TTA CCC GCT GGC (Seq ID no 85) GTCTTA TCG TCC TGC (Seq ID no 90) GTC TTC TCG TCC TGC (Seq ID no 91) GTGTTG TCG TCC TGC (Seq ID no 92) GTC TAT TCG TCC TGC (Seq ID no 93) GTCTCT TCG TCC TGC (Seq ID no 94) GTC TGT TCG TGC TGC (Seq ID no 95) AACACT CCC TTT GGA (Seq ID no 123) CAT GTG TCC TGT GGT (Seq ID no 117) CGTCAG CCC GAG AAA (Seq ID no 118) CAC TAC ACA CGC TCG, (Seq ID no 119) TGGCGT TGA GGT TTC (Seq ID no 120)

and a mixture of such probes.
 24. Peptide nucleic acid probes accordingto claim 22 selected from Lys(Flu)-Lys(Flu)-TCA CCA CCC TCC TCC-NH₂ (OK446/modified Seq ID no 6) Lys(Flu)-Lys(Flu)-CCA CCC TCC TCC-NH₂ (OK575/modified Seq ID no 6) Lys(Flu)-Lys(Flu)-ACT ATT CAC ACG CGC-NH₂ (OK447/modified Seq ID no 8) Lys(Flu)-ACT ATT GAG ACG CGC-NH₂ (OK688/modified Seq ID no 8) Lys(Flu)-Lys(Flu)-CCA CAC CCA CCA CAA-NH₂ (OK448/modified Seq ID no 12) Lys(Flu)-Lys(Flu)-AAC TCC ACA CCC CCG-NH₂ (OK449/modified Seq ID no 16) Lys(Flu)-Lys(Flu)-ACT CCA CAC CCC CGA-NH₂ (OK309/modified Seq ID no 17) Lys(Flu)-Lys(Flu)-ACT CCG CCC CAA CTG-NH₂ (OK450/modified Seq ID no 22) Lys(Flu)-Lys(Flu)-CTG TCC CTA AAC CCG-NH₂ (OK305/modified Seq ID no 23) Lys(Flu)-Lys(Flu)-TTC GAG GTT AGA TGC-NH₂ (OK306/modified Seq ID no 24) H-Lys(Flu)-TTC GAG GTT AGA TGC-NH₂ (OK682/modified Seq ID no 24) Lys(Flu)-Lys(Flu)-GTC CCT AAA CCC GAT-NH₂ (OK307/modified Seq ID no 25) Lys(Flu)-GTC CCT AAA CCC GAT-NH₂ (OK654/modified Seq ID no 25) H-Lys(Flu)-GAC CTA TTG AAC CCG-NH₂ (OK660/modified Seq ID no 29) Lys(Flu)-Lys(Flu)-Gly-GCA TCC CGT GGT CCT-NH₂(OK 223/modified Seq ID no 33) Lys(Flu)-Lys(Flu)-CAC AAG ACA TGC ATC-NH₂(OK 310/modified Seq ID no 34) Lys(Flu)-CAC AAG ACA TGC ATC-NH₂ (OK655/modified Seq ID no 34) H-Lys(Flu)-GGC TTT TAA GGA TTC-NH₂ (OK689/modified Seq ID no 40) H-Lys(Rho)-GGC TTT TAA GGA TTC-NH₂ (OK689/modified Seq ID no 40) Flu-β-Ala-β-Ala-GAT CAA TGC TCG GTT-NH₂ (OK624/modified Seq ID no 44) Flu-β-Ala-β-Ala-CGA CTC CAC ACA AAC-NH₂ (OK612/modified Seq ID no 76) Flu-β-Ala-β-Ala-GCA TTA CCC GCT GGC-NH₂ (OK623/modified Seq ID no 85) Lys(Flu)-GTC TTT TCG TCC TGC-NH₂ (OK745/modified Seq ID no 89) Lys(Rho)-GTC TTA TCG TCC TGC-NH₂ (OK746/modified Seq ID no 90) Lys(Rho)-GTC TTC TCG TCC TGC-NH₂ (OK746/modfied Seq ID no 91) Lys(Rho)-GTC TTG TCG TCC TGC-NH₂ (OK746/modified Seq ID no 92) Lys(Rho)-GTC TAT TCG TCC TGC-NH₂ (OK747/modified Seq ID no 93) Lys(Rho)-GTC TCT TCG TCC TGC-NH₂ (OK747/modified Seq ID no 94) Lys(Rho)-GTC TGT TCG TCC TGC-NH₂ (OK747/modified Seq ID no 95) Lys(Flu)-AAC ACT CCC TTT GGA-NH₂ (OK749/modified Seq ID no 123)

wherein Flu denotes a 5-(and 6)-carboxyfluoroescein label and Rhodenotes a rhodamine label, and a mixture of such probes.
 25. Use of apeptide nucleic acid probe according to claim 1 or a mixture thereof fordetecting a target sequence of one or more mycobacteria optionallypresent in a sample.
 26. Use of a peptide nucleic acid probe or amixture thereof according to claim 25 for detecting a target sequence ofone or more mycobacteria of the Mycobacterium tuberculosis Complex(MTC), in particular a target sequence of M. tuberculosis.
 27. Use of apeptide nucleic acid probe or a mixture thereof according to claim 25for detecting a target sequence of one or more mycobacteria other thanmycobacteria of the Mycobacterium tuberculosis Complex, in particular atarget sequence of one or more mycobacteria of the Mycobacterium aviumComplex.
 28. Method for detecting a target sequence of one or moremycobacteria optionally present in a sample comprising (1) contactingany rRNA or rDNA present in said sample with one or more peptide nucleicacid probes according to claim 1 or a mixture thereof under conditions,whereby hybridisation takes place between said probe(s) and said rRNA orrDNA, and (2) observing or measuring any formed detectable hybrids, andrelating said observation or measurement to the presence of a targetsequence of one or more mycobacteria in said sample.
 29. Methodaccording to claim 28 for detecting a target sequence of one or moremycobacteria of the Mycobacterium tuberculosis Complex (MTC), inparticular a target sequence of M. tuberculosis.
 30. Method according toclaim 28 for detecting a target sequence of one or more mycobacteriaother than mycobacteria of the Mycobacterium tuberculosis Complex. 31.Method according to claim 28, wherein the hybridisation takes place insitu.
 32. Method according to claim 28, wherein the hybridisation takesplace in vitro.
 33. A method according to claim 28, characterised inthat a signal amplifying system is used for measuring the resultinghybridisation.
 34. Method according to claim 28, wherein the sample is asputum sample.
 35. Kit for detecting a target sequence of one or moremycobacteria, in particular a target sequence of one or moremycobacteria of the Mycobacterium tuberculosis Complex (MTC), inparticular a target sequence of M. tuberculosis, and/or for detecting atarget sequence of one or more mycobacteria other than mycobacteria ofthe Mycobacterium tuberculosis Complex (MOTT), in particular a targetsequence of one or more mycobacteria of the Mycobacterium avium Complex,characterised in that said kit comprises at least one peptide nucleicacid probe according to claim 1, and optionally a detection system withat least one detecting reagent.
 36. Kit according to claim 35,characterised in that it further comprises a solid phase capture system.